Difference between revisions of "Team:UofC Calgary/Experiments"

Latest revision as of 01:05, 20 October 2016

iGEM Calgary 2016

Experiments

BioTarget

Double-Stranded Break Assay using Immunofluorescence
Experimental Details and Rationale	Exposure to ionizing radiation causes DNA double strand breaks (DSBs) in cells, which are naturally repaired by cells over time. DSBs in nuclear DNA can be visualized in cells by immunofluorescence staining of DSB markers – namely 53BP1, a protein which binds near DSBs. As a result, nuclear foci form, each corresponding to a DSB. Over time, the number of foci are reduced due to DNA repair. To test for radioprotection by mBBI, we wanted to study the repair kinetics in cells treated with mBBI against a control. Cells were grown to confluency, irradiated and fixed at various time points over 24 hours. Immunofluorescence staining was performed to visualize 53BP1 and the resulting foci were counted.
Materials	Glass coverslips and microscopy slides 1BR3 primary fibroblast cells in Modified Eagle Medium (MEM) - (10^5 cells/ml) 30 mm Cell Culture Plates 1x PBS 1x PBS solution with 3% PFA (w/v) and 2% (w/v) sucrose 1x PBS solution with 2% (w/v) Bovine Serum Albumin 1x PBS solution with 0.2% (v/v) Triton X100 1x PBS solution with 0.1μg/ml 4’,6-diamidino-2-phenylindole (DAPI) Mouse anti-53BP1 Antibody Rabbit anti-H2AX Antibody Cy3 anti-Mouse Antibody FITC anti-Rabbit Antibody Mounting medium for fluorescence (Flouromount G or Vectashield)
Protocol	Prep and Irradiation Plate 2 mLs of 1BR3 cells at the density of 10^5 cells/ml MEM into 22 30 mm plates containing glass coverslips. Incubate cells until 100% confluency is observed on the glass coverslips using a brightfield microscope. Treat the confluent plates with mBBI (as outlined in Table 1) and incubate at 37℃ with 5% CO for 6 hours. Irradiate plates which are intended for irradiation (outlined in Table 1) with 2 Gy. We used a Gammacell 1000 irradiator (MDS Nordion). Immunofluorescence Staining When plates reach their intended time point, remove the media and wash in 1X PBS. Perform washes by pouring PBS into the dish beside the coverslip, with special care not to pour media directly onto the coverslips, which could dislodge the cells. All washes hereon are done in this manner. Cells on glass coverslips are fixed for 10 minutes by adding 100 mL of 1X PBS solution with 3% PFA (w/v) and 2% sucrose (w/v) to the top of the coverslip, forming a dome on the coverslip. Wash cells twice in 1X PBS and store in 1X PBS until all plates have been fixed at their corresponding time points. Add 100 mL of 1X PBS solution with 0.2% Triton X100 (v/v) to the cells for 3 minutes to allow permeabilization. Incubate cells in 100 mL of a 1:800 dilution of Mouse anti-53BP1 and Rabbit anti-H2AX in 2% BSA for 1 hour. Pipet the solution dropwise onto the coverslips. Wash cells three times in 1X PBS. 100 μl of secondary antibodies Cy3 anti-Mouse and FITC anti-Rabbit pipetting dropwise onto the coverslips. Cells are incubated in this solution for 20 minutes in the dark, because FITC and Cy3 are sensitive to light and lose their fluorescence if prematurely exposed. Wash cells 3 times in 1X PBS. Pour 2 mls of 1X PBS containing 0.1 mg/mL DAPI into the dishes and leave on for 10 mins. Wash cells 3 times in 1X PBS. Microscopy slides were prepared by adding one drop of Vectashield onto the slide. Carefully lift coverslips from the cell culture dishes using a razor blade or sharp edged inplement and plate onto microscopy slides such that the cell bed (top of the coverslip) is placed face down on the slide. Seal the edges of the coverslip onto the slide by brushing on clear nail polish over the edges. Counting Foci Randomize cells by using opaque tape to cover their labels and get them randomly assorted by another lab member. Number them on the tape for ease of reference when counting. Foci can be counted on an epifluorescent microscope under red and green channels.

Table 1: Experimental Outline of mBBI treatment and Ionizing Radiation. mBBI treatment was administered at 30uM and cells were irradiated at 2 Gy.

Clonogenic Cell Survival Assay
Experimental Details and Rationale	Ionizing radiation is capable of killing cells by damaging their DNA, causing single and double strand breaks, which trigger apoptosis. If the DNA damage can be repaired, the cells survive. If not, they will die. To test whether mBBI elicits radioprotection, the survival of cells can be assessed. The purpose of this assay is to assess the viability of cells when treated with ionizing radiation, with and without mBBI.
Materials	MEM media with 15% Fetal Bovine Serum (v/v) and 10% Penicillin-Streptomycin (v/v) T-75 flasks 60 mm Cell culture plates 1BR3 cells Trypan Blue Dye 0.25% Trypsin EDTA 15ml Centrifuge Tube
Protocol	Preparation of Cell Lines Prior to Setting up Clonogenic Cell Survival Label 6 T-75 flasks with a known number of cells and radiation in Gy (0 and 5). Add 5 ml of growth medium to the flasks and keep them in a biosafety hood. Trypsinize the stock flask of cells to be tested for radiosensitivity. Obtain a cell count, then add 250,000 cells to the 5ml medium in the T-75 flask. Place T-75 flasks with the cell cultures in a 37°C incubator with 5% CO₂, and keep cap to the flask loose to allow gas exchange. Allow the cells to settle and attach as a monolayer. Irradiation of Flasks and Performance of Plating Experiment for Clonogenic Assay Label all 60 mm plates according to cell line, drug and radiation exposure. Place 5mL of complete medium in each plate Label all 15 ml tubes according to 1:1, 1:10, 1:100 for each Gy dose level (0 and 5). Put 4.5 ml of complete medium into the 1:10 and 1:100 tubes, but not the 1:1 tubes. Ensure that the Coulter counter is set to the appropriate size parameter for the cell line. Irradiate the flasks at the appropriate dose. Starting with the 0 Gy flask, aspirate the medium and then rinse the cells with PBS, then trypsinize. Place these harvested cells in the 15ml tube labelled as 0 Gy, 1:1. Take 100 ul of the 0 Gy, 1:1 solution and place in the counting vial for 0 Gy. Now aspirate, rinse cells with PBS and trypsinize the next flask, 5 Gy. While the 5 Gy flask is on the warming tray, count the 0 Gy counting tray using the Coulter counter and record this data. As before, place the harvested 5 Gy cells into the 15 ml tube labelled 5 Gy, 1:1. Take 100 ul of the 5 Gy 1:1 solution and put into the 5 Gy counting vial. Use a Coulter counter to count cells as before. Serial Dilutions and Incubation Resuspend the cell pellet of the 15 ml tube. Place 0.5 ml of the 1:1 dilution into the 1:10 dilution tubes of both the 0 Gy and 5 Gy tubes. Calculate the volume of the cell solution needed to plate ~50-100 cells. Place this volume of the appropriate dilution (probably 1:100) onto the appropriate 60 mm plate. Spread this medium drop by drop, ensuring that the plate is covered equally. You can shake the plate to distribute the culture evenly, but do not swirl. Only back-and-forth motions. Place all plates into the incubator. Incubate for 14 days at 37C with 5% CO2. Staining of Plates Take out all plates from the incubator and aspirate media with vacuum Add 1mL fixing solution to each of the plates (3% acetic acid, 8% methanol, 89% dH2O) Allow cells to sit in fix solution for 2 minutes, then aspirate fix solution Add 1mL stain solution to each of the plates (0.2% (w/v) Gentian violet, 10% formalin in PBS) Let plates sit in stain solution for 5 minutes and aspirate stain solution Add dH₂O at room temperature to the plates to de-stain excess Gentian Violet. Repeat twice, or until only the colonies are stained as opposed to the plate. Let the plates air-dry overnight. Count the next day. Counting of Colonies Take the air-dried plates and place in ColCount machine after blanking the machine. Count using small colony reference program and record data in a table. Calculate plating efficiency (PE) by finding the average colony counts for the 3 plates and dividing that by the number of cells plated, then multiply by 100%. ((Ave colonies counted / Number of cells plated) X 100 %). To find the the surviving fraction (SF), take the PE of the treated sample and divide that by the PE of the control, then multiply by 100%. ((PE treated / PE control) X 100%)

CHASSIS

Rehydration of Registry DNA
Experimental Details and Rationale	We used standard parts from the iGEM registry to ligate our synthesized genetic constructs into the pSB1c3 backbone. We also used BBa_J04450 (pSB1c3 backbone containing RFP) as a positive control to confirm transformation of E. coli and B. subtilis.
Materials	iGEM 2016 distribution kit ddH₂O
Protocol	Add 10 uL ddH₂O to desired well (it will become red). Pipette up and down. Incubate at room temperature for 10 minutes. Transform cells with 1 μL of DNA. Store at -20°C.

Rehydration of IDT Synthesized DNA
Experimental Details and Rationale	We ordered genetic constructs from IDT to clone into the pSB1c3 backbone.
Materials	Synthesized DNA from IDT ddH₂O
Protocol	Centrifuge tube containing DNA for 3-5 seconds at 3000 g, ensuring all material is at the bottom of the tube. Add ddH2O to reach a final concentration of 50 ng/μL. Vortex. Incubate at 50°C for 20 minutes. Vortex and centrifuge. Store at -80°C.

Plasmid MiniPrep from Escherichia coli and Bacillus subtilis
Experimental Details and Rationale	Plasmids were amplified in E. coli cells in order to use them for confirmation of ligation and transformation of B. subtilis.
Materials	2.5 mL overnight culture of transformed bacteria in appropriate antibiotic in 16x125 mm culture tube Resuspension buffer (store at 4°C) 50 mM Tris-HCl ,pH 8 10 mM EDTA 100 μg/mL RNase A Lysis buffer 200 mM NaOH 1% (v/v) SDS Precipitation buffer 3 M CH3CO2K, pH 5.5 Isopropanol 70% ethanol Table-top centrifuge 2 mL microcentrifuge tubes 1.5 mL microcentrifuge tubes
Protocol	Grow 2.5 mL of transformed culture overnight in Luria-Bertani broth with appropriate antibiotic. Transfer 2 mL to a 2 mL microcentrifuge tube and pellet the cells by spinning at 3500 g for 1 minute. Discard supernatant. Resuspend pellet in 300 μL Resuspension buffer. Add 300 μL Lysis buffer, invert gently and incubate at room temperature for 3-5 minutes. Add 300 μL Precipitation buffer, invert gently. A white precipitate should form. Centrifuge at 14,000 g for 10 minutes at room temperature. Retain supernatant in a clean 1.5 mL microcentrifuge tube. Add 650 μL isopropanol, gently invert and incubate at room temperature for 10 minutes. Centrifuge at 14,000 g for 10 minutes at 4°C. Discard supernatant. Wash pellet with 500 μL cold 70% ethanol. Add to microcentrifuge tube, do not resuspend. Centrifuge at 14,000 g for 5 minutes at 4°C. Discard supernatant. Dry pellet in speed vac for 10 minutes. Resuspend pellet in ddH₂O and store at -20°C.

Restriction Digest
Experimental Details and Rationale	Restriction digests were performed on plasmid backbones and synthesized DNA inserts before ligating them together, as well as to confirm ligation and transformation of cells with desired plasmids.
Materials	DNA Restriction enzymes 100X Bovine Serum Albumin (BSA) 10X appropriate buffer ddH₂O 0.2 mL PCR tubes
Protocol	Into a 0.2 mL PCR tube, add the following: 1 μg DNA 1 μL 100X BSA 1 μL restriction enzyme 1 1 μL restriction enzyme 2 2 μL 10X appropriate buffer ddH₂O to a total volume of 20 μL Incubate tube at 37°C for 1 hour. Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes.

Antarctic Phosphatase Treatment
Experimental Details and Rationale	Vectors were treated with Antarctic phosphatase to remove their 5’-phosphate group and prevent them from self-ligating before addition of the digested insert.
Materials	Digested DNA vector Antarctic phosphatase buffer Antarctic phosphatase ddH₂O 0.2 mL PCR tubes
Protocol	To vector tube from restriction digest, add: 1 μL 10X Antarctic phosphatase buffer 1 μL Antarctic phosphatase 8 μL ddH₂O Incubate tube at 37°C for 30 minutes. Deactivate Antarctic phosphatase via heat shock by incubating tube at 80°C for 20 minutes.

Ligation of DNA Inserts to Plasmid Backbones
Experimental Details and Rationale	Synthesized DNA inserts were ligated into plasmid backbones for propagation and use in E. coli and B. subtilis as well as for submission to the registry.
Materials	Digested vector DNA Digested insert DNA 10X DNA ligase buffer (from New England Biolabs) T4 DNA ligase (1 U/μL) (from New England Biolabs) ddH₂O 0.2 mL PCR tubes
Protocol	To a 0.2 mL PCR tube, add: 50 ng digested vector DNA appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector 1 μL T4 DNA ligase 2 μL 10X T4 DNA ligase buffer ddH₂O to a total volume of 20 μL Incubate tube at room temperature overnight. Use 10 μL to transform cells, store at -20°C.

Colony PCR for Escherichia coli and Bacillus subtilis using the KAPA HiFi PCR Kit
Experimental Details and Rationale	Colony PCR was used to confirm the presence of plasmids with desired ligated insert in transformed E. coli and B. subtilis >cells.
Materials	Transformed bacterial colony on agar plate 10X Taq polymerase buffer Taq DNA polymerase (% U/μL) 10X Buffer A 10 mM dNTP 10 μM forward primer 10 μM reverse primer PCR-grade ddH₂O 0.2 mL PCR tubes
Protocol	Sample Preparation Add 4 μL of PCR-grade ddH₂O to 0.2 mL PCR tube. Using aseptic technique, pick a colony and touch it with a sterile pipette tip and place it in the PCR tube for 5-10 seconds. To each PCR tube, add: 0.2 μL Taq DNA polymerase 5 μL 10X Buffer A 1 μL 10 mM dNTP 2 μL 10 μM forward primer 2 μL 10 μM reverse primer 8 μL PCR-grade ddH₂O Running PCR Run PCR in a thermal cycler under the following conditions: Initial denaturation: 95°C for 3 minutes Denature: 95°C for 30 seconds Anneal: Tm-5°C for 30 seconds Extension: 72°C for 1 minute per kilobase Repeat denature, annealing, extension steps for 30-35 cycles Final extension: 72°C for 5 minutes

Preparation of Agar with Antibiotics
Experimental Details and Rationale	Various antibiotics (kanamycin, chloramphenicol, ampicillin and hygromycin) were used to select for successful E. coli and B. subtilis transformants.
Materials	Luria-Bertani broth with agar 10% (w/v) tryptone 5% (w/v) NaCl 10% (w/v) yeast extract 15% (w/v) agar Appropriate antibiotic kanamycin (final concentration of 100 μg/mL) chloramphenicol (final concentration of 30 μg/mL) ampicillin (final concentration of 50 μg/mL) hygromycin (final concentration of 100 μg/mL) 1500 mL Erlenmeyer flask
Protocol	In a 1500 Erlenmeyer flask, add 10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar in 1000 mL dH₂O. Dissolve solids and make sure to add a stir bar. Cover flask loosely with aluminum foil, secure with autoclave tape, and sterilize by autoclaving for 20 minutes. Remove agar from autoclave using oven mitts. Allow agar to cool until warm to the touch before adding appropriate antibiotic. Stir on hot plate and magnetic stirrer for 30 seconds. Pour agar into plates using aseptic technique.

Preparation of Chemically Competent Escherichia coli Cells
Experimental Details and Rationale	Competent E. coli cells were transformed with ligated plasmid to amplify them for further use in digest confirmation and B. subtilis transformation.
Materials	Escherichia coli TOP10 cells Luria-Bertani broth 10% (w/v) tryptone 5% (w/v) NaCl 10% (w/v) yeast extract 16x125 mm culture tubes 250 mL Erlenmeyer flask Spectrophotometer Centrifuge 50 mL Falcon tubes 50 mM CaCl2 50 mM CaCl2, 15% glycerol 1.5 mL microcentrifuge tubes
Protocol	Inoculate 5-10 mL of Luria-Bertani broth with E. coli TOP10 cells and allow it to incubate at 37°C overnight, shaking at 200 rpm. Subculture 1 mL of E. coli overnight culture in 49 mL fresh Luria-Bertani broth in a 250 Erlenmeyer flask. Incubate at 37°C, shaking at 200 rpm until it reaches an OD₆₀₀ of 0.4-0.6 (usually takes 2.5 hours). Spin down cells in 50 mL Falcon tube at 8200 g for 10 minutes at 4 °C. Resuspend cells in 12.5 mL cold 50 mM CaCl₂ and place on ice for 10 minutes. Repeat Step 3. Resuspend cells in 2 mL cold 50 mM CaCl₂, 15% glycerol and place on ice for 30 minutes. Separate cells into aliquots of 200 μL and store at -80°C.

Glycerol Stock Preparation of Transformed Escherichia coli and Bacillus subtilis
Experimental Details and Rationale	Glycerol stocks of transformed E. coli and B. subtilis were prepared for long-term storage and future use.
Materials	Overnight culture of transformed bacteria Sterile 1.5 mL cryo-tubes Sterile 50% glycerol
Protocol	Using aseptic technique, pipette 0.5 mL of 50% sterile glycerol into a 1.5 mL cryo-tube. Using aseptic technique, add 0.5 mL of overnight culture. Pipette up and down gently to mix. Store at -80°C for up to 1 year.

Plating Culture Broth on Agar Plates
Experimental Details and Rationale	Culture broth was plated on agar to isolate single colonies.
Materials	Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria 70% ethanol Spreading rod Bunsen burner
Protocol	Using aseptic technique, pipette 50-100 μL of bacterial culture onto antibiotic agar plate. Dip spreading rod in 70% ethanols, pass over flame and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time. Dip spreading rod in 70% ethanol, pass over flame and allow for excess liquid to burn off. Incubate plates at 37°C overnight or until growth is observed.

Streaking of Agar Plates
Experimental Details and Rationale	Culture broth was streaked on agar plates to isolate single colonies.
Materials	Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria or single isolated colony on agar plate Inoculation loop Bunsen burner
Protocol	Using aseptic technique, flame inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar. Dip the inoculation loop in bacterial culture or touch a single colony and streak the loop on ¼ of the surface of agar in a zigzag motion. Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar. Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1.4 of the surface of the agar. Repeat Steps 3 and 4 two more times. Flame the inoculation loop until red hot. Allow it to cool for 10 seconds. Incubate plates at 37°C overnight or until growth is observed.

Agarose Gel Electrophoresis
Experimental Details and Rationale	Digested and undigested plasmids were run on agarose gels to confirm the presence and proper orientation of DNA inserts.
Materials	TAE buffer 40 mM Tris, pH 7.6 20 mM CH₃COOH 1 mM EDTA Agarose 250 mL Erlenmeyer flask RedSafe Nucleic Acid Staining Solution Gel casting tray and comb 10X loading dye DNA sample Microwave
Protocol	For a 1% gel (standard), add 1 g agarose to 100 mL TAE buffer in a 250 mL Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long). Allow flask to cool in fumehood until warm to the touch before adding 5 μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix. Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer. Load samples of 20 μL DNA containing 2 μL loading dye. Run gel at 120 V for 45 minutes or until loading dye is ⅔ way down the gel.

Growth Curve of Bacillus subtilis: Temperature Simulation
Experimental Details and Rationale	To simulate the temperature conditions of B. subtilis in our patch, we measured bacterial growth in 10 mL of Luria-Bertani (LB) broth over the course of 24 hours at varying temperatures of 35°C (average skin temperature), 22°C (average room temperature on board the International Space Station) and 4°C (negative control). Growth was measured by spectrophotometer at a wavelength of 600 nm (OD₆₀₀).
Materials	Luria-Bertani broth medium with hygromycin 10% (w/v) tryptone 5% (w/v) NaCl 10% (w/v) yeast extract Final concentration of 100 μg/mL hygromycin Bacillus subtilis WB800 cells 16x125 mm culture tubes 50 mL Falcon tubes Spectrophotometer 1 mL cuvette
Protocol	*Preparation of B. subtilis* WB800 Cells** Two days prior to measuring growth, prepare an overnight culture of B. subtilis in 10 mL LB broth with the appropriate antibiotic (100 μg/mL final concentration of hygromycin). Incubate at 37°C, shaking at 200 rpm overnight. Inoculation One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35°C, 22°C or 4°C) with lids loosened, shaking at 200 rpm. Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement. Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement. Measurement of Growth Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD₆₀₀). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once! Commence measurement of samples at 8:00 (OD₆₀₀). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50. To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD₆₀₀ >0.4, dilute the sample 10X using LB-broth with 100 μg/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.

Growth Curve of Bacillus subtilis: Media Optimization
Experimental Details and Rationale	Our patch will contain three attached packets of additional media that can be ruptured to extend the cell growth and production of mBBI. In order to determine which media would be best suited for these packets, we tested three different types of media: Luria-Bertani broth, 2X Luria-Bertani broth, and Super Rich broth. We added 1 mL of these three types of media to 9 mL cultures of B. subtilis WB800 in LB broth containing 100 μg/mL hygromycin every 12 hours and measured bacterial growth over the course of 72 hours. Growth was measured by spectrophotometer at a wavelength of 600 nm (OD₆₀₀).
Materials	Luria-Bertani broth medium with hygromycin 10% (w/v) tryptone 5% (w/v) NaCl 10% (w/v) yeast extract Final concentration of 100 μg/mL hygromycin 2X Luria-Bertani broth medium with hygromycin 20% (w/v) tryptone 10% (w/v) NaCl 20% (w/v) yeast extract Final concentration of 100 μg/mL hygromycin Super Rich broth medium 2.5% (w/v) tryptose 2% (w/v) yeast extract 0.3% K₂HPO₄ 3% glucose (v/v) Notes: Mix all components except glucose and adjust pH to 7.5 Autoclave solution except for glucose. Add 1 mL/L of anti-foam prior to autoclave. Filter-sterilize glucose (20%) and add to main solution to a final concentration of 3%. Adapted from Haling et al., 1997 Bacillus subtilis WB800 cells 16x125 mm culture tubes 50 mL Falcon tubes Spectrophotometer 1 mL cuvette
Protocol	*Preparation of B. subtilis* WB800 Cells** Two days prior to measuring growth, prepare an overnight culture of B. subtilis in 10 mL LB broth with the appropriate antibiotic (100 μg/mL final concentration of hygromycin). Incubate at 35°C, shaking at 200 rpm overnight. Inoculation One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at 37°C with lids loosened, shaking at 200 rpm. Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement. Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement. Measurement of Growth Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD₆₀₀). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once! Commence measurement of samples at 8:00 (OD₆₀₀). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50. To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD₆₀₀ >0.4, dilute the sample 10X using LB-broth with 100 μg/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to incubate at 35°C with lids loosened, shaking at 200 rpm. Add 1 mL of appropriate media (either LB, 2XLB or Super Rich) to each 50 mL Falcon tube every 12 hours for a total of three times (at 12 hours, 24 hours and 36 hours) after inoculation.

Transformation of Escherichia coli TOP10
Experimental Details and Rationale	Escherichia coli TOP10 cells were used to amplify newly-ligated plasmids and were later harvested for future use in digest confirmation and Bacillus subtilis transformation.
Materials	Competent E. coli TOP10 aliquots (200 μL) DNA for transformation Luria-Bertani broth 10% (w/v) tryptone 5% (w/v) NaCl 10% (w/v) yeast extract Agar plate with appropriate antibiotic
Protocol	Thaw 200 μL aliquot of competent E. coli TOP10 cells on ice just before use. Add 0.3-1 μg DNA to cells (in maximum 20 μL), flick gently to mix, and place on ice for 30 minutes. Heat shock for 60-75 seconds at 42°C. Place on ice for 5 minutes. Add 250 μL Luria-Bertani medium to aliquot of cells. Incubate cells for 60 minutes at 37°C, shaking at 200 rpm for 1 hour. Pellet cells in a microcentrifuge at 3500 g for 1 min and discard supernatant. Resuspend pellet in 250 μL Luria-Bertani broth. Plate 50-100 μL of resuspended culture on agar plate with appropriate antibiotic and spread. Incubate plates at 37°C overnight or until desired growth is observed.

Preparation of SP1 and SP2 Media for Bacillus subtilis WB800 Transformation (adapted from Spizizen, 1958)
Experimental Details and Rationale	Bacillus subtilis was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.
Materials	SP1 Salts >0.2% (w/v) (NH₄)₂SO₄ 1.4% (w/v) K₂HPO₄ 0.6% (w/v) KH₂PO₄ 0.1% Na₃C₆H₅O₇⋅2H₂O 0.02% MgSO₄⋅7H₂O 50 mM CaCl₂ 250 mM MgCl₂ 50% filter-sterilized glucose Casamino acids/yeast extract 2% (w/v) casamino acids 10% yeast extract
Protocol	For all stock solutions, autoclave solutions for 20 minutes and store at room temperature. To make SP1 media, add any volume of SP1 salts plus 1/100 volume glucose, 1/100 volume casamino acids/yeast extract, and 0.5% (w/v) tryptophan. To make SP2 media, add 1/100 volume of CaCl₂ and 1/100 volume of MgCl₂ to any volume of SP1 media.

Preparation of Bacillus subtilis WB800 Competent Cells and Transformation
Experimental Details and Rationale	B. subtilis was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.
Materials	Luria-Bertani agar plate with appropriate antibiotic 16x125 mm culture tubes 13x100 mm culture tubes Sterile SP1 Medium Sterile SP2 Medium 100 mM EGTA DNA for transformation
Protocol	Streak cells on Luria-Bertani agar plate in the evening at 17:00. Incubate cells at 30°C overnight. At 9:00 the next morning, transfer cells to 2 mL SP1 medium in 16x125 mm culture tubes. The culture should be slightly turbid. Incubate at 37°C for 3 hours and 45 minutes, shaking at 300 rpm. Transfer 0.5 mL SP1 culture to 4.5 mL SP2 medium pre-warmed to 37°C in a 16x125 mm culture tube. Incubate at 37°C for 1 hour and 30 minutes, shaking at 150 rpm. Add 50 μL 100X EGTA to the SP2 culture and incubate at 37°C for another 10 minutes, shaking at 150 rpm. Take 0.5 mL competent cells and transfer to a sterile 13x100 mm culture tube. Add 0.1-3 g DNA in a volume of 60 μL or less. Incubate at 37°C for 1 hour and 30 minutes, shaking at 300 rpm. Plate 0.25 mL of sample on Luria-Bertani agar plate with appropriate antibiotic. Incubate plate at 37°C overnight or until desired growth is observed.

DEVICE

Semipermeable Membrane Diffusion Assay
Experimental Details and Rationale	The purpose of this assay was to determine quantitatively if the membrane in our transdermal patch can act as a filter and prevent bacteria cells from diffusing through the semipermeable membrane.
Materials	50 mL falcon tubes 15 mL falcon tubes Distilled/non distilled water NaCl 20 mL overnight culture (B. subtilis WB800) LB media CoTran 9716 and 9728 semipermeable membrane Parafilm
Protocol	Preparation Prepare 500 mL of 0.9% w/v saline solution using sodium chloride at a pH of 7.35. Cover using parafilm and store at room temperature. Start 20 mL overnight culture B. subtilis. For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of B. subtilis. Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike. Repeat process using cell culture in distilled water. Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay. As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got. Measurements Aliquot 5 mL of each overnight culture into three 15 mL falcon tubes. With one of the 15 mL falcon tube wrap parafilm over the top of the tube. With the other, place the 9716 semipermeable membrane over the top of the tube and Parafilm around the edges to prevent it from falling off. Repeat using 9728 semipermeable membrane With the 50 mL falcon tubes, add 30 mL of saline solution Invert and submerse the 15 mL falcon tubes into the 50 mL falcon tubes. Cover the top of the 50 mL falcon tube with Parafilm. After a 24 hour period take a 1 mL sample of the water in the 50 mL falcon tube. Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm. Repeat once daily for seven days.

Filter Sterilization Membrane Diffusion Assay
Experimental Details and Rationale	In running the first diffusion assay, our data analysis showed that there was an increased amount of cell detection, meaning that the cells may or may not be diffusing through. However, we suspected that there might be contamination so we repeated the assay with a few changes. This time, we are using a filter sterilization membrane that has pore sizes of 0.2 micron. We are also adding hygromycin to the saline solution for B. subtilis WB800 and chloramphenicol for E.coli mBBI-GFP. This ensured that there would be no contamination in our samples.
Materials	10 mL syringes Filter sterilization membrane Distilled/non distilled water NaCl Hygromycin (20 mg/mL) 20 mL overnight culture (B. subtilis WB800) LB media 250 mL erlenmeyer flasks Chloramphenicol agar plates
Protocol	Preparation Prepare 500 mL of 0.9% W/V saline solution using sodium chloride at a pH of 7.35. Add appropriate antibiotics. Cover using parafilm and store at room temperature. Start 20 mL overnight culture B. subtilis. For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of B. subtilis. Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike. Repeat process using cell culture in distilled water. Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay. As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got. Measurements Take up 5 mL of each overnight culture into two 10 mL syringes. Take up 5 mL chloramphenicol LB media into one 10 mL syringe. Attach filter sterilization membrane to one of the syringes containing the 5 mL overnight culture and to the syringe containing 5 mL chlor + LB media. With the 250 mL erlenmeyer flasks, add 10 mL of saline solution. Submerse each of the 10 mL filter sterilization syringes into the 250 mL erlenmeyer flasks. Cover the top of the erlenmeyer flasks with Parafilm. After a 24-hour period take a 1 mL sample of the saline solution in the 250 mL erlenmeyer flasks. Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm. Take a 150 µL sample of the saline solution and plate it on respective antibiotic plate. Repeat once daily for seven days.

Backing Layer Growth Curve Assay
Experimental Details and Rationale	The purpose of this assay was to determine quantitatively if the backing layer will allow cell growth similar to those found under optimal conditions.
Materials	10 mL culture tube CoTran 9722 backing layer 50 mL falcon tubes LB media with hygromycin Hygromycin agar plates
Protocol	*Preparation of B. subtilis* WB800 Cells** Two days prior to measuring growth, prepare an overnight culture of B. subtilis in 10 mL LB broth with the appropriate antibiotic (100 μg/mL final concentration of hygromycin). Incubate at 37°C, shaking at 200 rpm overnight. Inoculation One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35°C, 22°C or 4°C) with lids loosened, shaking at 200 rpm. Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement. Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement. Measurement of Growth Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD₆₀₀). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once! Commence measurement of samples at 8:00 (OD₆₀₀). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50. To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD₆₀₀ >0.4, dilute the sample 10X using LB-broth with 100 μg/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.

Backing Layer Survival Assay
Experimental Details and Rationale	Following the first backing layer growth curve assay, we consulted our mentors and determined that the assay was not effective in determining whether our backing layer would be a limiting factor for gas exchange. This is due to the fact that there is 10x the volume of oxygen in the culture tube to begin with. Therefore, the rate of gas exchange through the backing layer will be negligible. This purpose of this assay was to determine whether the bacterial cells can be starved of oxygen with our backing layer using a different protocol.
Materials	10 mL culture tube 2x2 well nuclon plates CoTran 9722 backing layer CoTran 9719 backing layer Parafilm
Protocol	Day Zero Using sterilized 2x2 culture plates, add 1.62 mL of LB media + appropriate antibiotic. Add 180 µL O/N culture that has already been OD₆₀₀. This will be the baseline reading. Pipette 180 µL of the mixture out, discard. Place appropriate backing layer on top of the culture plates. Secure backing layer with Parafilm on top criss cross and gain around the edges. For a positive control, cover culture plate with Parafilm. Puncture a hole into each of the wells using a syringe needle. For a negative control, place the lid on top and Parafilm to secure. Place in incubator at 35°C at 10 rpm. Repeat for replicates. Day One Begin OD₆₀₀ readings using LB + antibiotics. Take the reading of the day one plates. Repeat for all membranes and controls Day Three Repeat day one procedure using day three plates Day Seven Repeat day one procedure using day seven plates

in vivo Mouse Testing
Ethics	With the tireless support of Dr. Craig Jenne, the team wrote and submitted an extensive ethics application to the Health Sciences Animal Care Committee (HSACC) which evaluated the application on multiple grounds such as feasibility, morality, precautions, etc. of the testing of our transdermal patch on mice models. Our protocols and the scientific significance of our transdermal patch were reviewed and approved by the HSACC.
Experimental Details and Rationale	This experiment allowed us to examine the effects of the prototype version of our transdermal patch in vivo. This experiment addresses three main aims: To determine whether patches containing TD1 tagged mBBI diffused from the patch into systemic circulation of mice. To determine whether the membrane of the patch was capable of containing B. subtilis without leakage. To determine whether the adhesive, the peptide or the B. subtilis is immunogenic. Three cohorts of six mice each were used for this experiment. The first cohort was given patches containing water to examine the effects of the adhesive on the skin. In the second cohort, mice were given a patch containing B. subtilis. The third group was used to examine the diffusion of fusion protein across the skin. In order to measure the effects of these patches, the skin samples were obtained post-euthanasia and tested for any immunological responses (ie. neutrophil deposition). The blood samples were collected to check for mBBI presence using mass spectrometry. The blood samples collected were either isolated for serum or for plasma. Since it was unknown where mBBI would be found, both procedures were conducted.
Materials	18 BALB/c adult mice 18 transdermal patches with respective contents General Anesthesia Hair removal cream 0.5 M EDTA
Protocol	Prepare patches with their respective contents in the protocol described here. Manufacture at least 6 working patches of each type. Use 6 mice for treatment, one for each patch type. Anesthetize mice with 300 uL of general anesthesia. The anesthesia was administered to 6 mice at a time.e. Remove hair using hair removal cream (Nair) underneath the shoulder of the mice. Apply one patch to one mouse on the top of lumbar vertebrae using tissue glue to ensure proper attachment. After the application, leave mice caged. Repeat Steps 3-5 for other mice in other patch groups. Keep mice under constant observation to ensure that patch stays on. After day 2, anesthetize three mice with 600uL of general anesthesia each. (Note: This amount of anesthesia is used when the mice will be sacrificed in the immediate future.) Once they are unconscious, draw blood samples two of the mice via the left ventricle to obtain blood. Leave the blood sample at room temperature for half an hour, and upon coagulation, isolate serum by centrifugation. Anesthetize the remaining mouse and collect samples using 8 uL EDTA in order to obtain blood plasma. Once the blood is mixed with the anticoagulant, the plasma is isolated by spinning down the sample. Sacrifice the mice via cervical dislocation. Obtain skin samples from the lumbar region. At day 5, repeat Steps 8-11.

Detection of mBBI by Mass Spectrometry
Experimental Details and Rationale	Mass spectrometry was used to determine the presence of mBBI in mouse trial blood samples.
Materials	Mouse blood sample Heavy isotope of mBBI peptide C18 Zip-Tip column from Thermo 50% acetonitrile 0.1% formic acid 14 cm C18 column Mass spectrometer
Protocol	Extract peptides from mouse blood plasma or serum via purification on a C18 Zip-Tip column from Thermo; elute with 50% acetonitrile, 0.1% formic acid. Concentrate samples 10x via evaporation and resuspend in 0.1% formic acid. Inject extracted samples onto a 10 cm C18 column and elute with a 30 minute 5-40%B gradient at 300 nL/min. Acquire MS spectra on a Thermo LTQ Orbitrap Velos in ion trap mode or in Orbitrap mode with CID. Analyze spectra manually for the presence of mBBI (monoisotopic mass = 2577.1 Da) at various charge states. Note: Pure sample of mBBI, synthesized by BioBasic, was used as a reference for identification.

Immunohistochemical Staining of Mouse Cryo Cross-Section
Experimental Details and Rationale	This technique was employed to study any possible immunological responses induced by the transdermal patch, the bacteria Bacillus subtilis, and mBBI. Mice skin tissue samples were obtained from 9 mice at day 2 (samples 1-3 for all treatments) and the rest of the mice samples were obtained on day 5 (samples 4-6 for all treatments) of the study. The skin tissue samples were then fixed, sectioned, and stained. Due to lack of training and materials, members of the Jenne lab had to perform most of the experiment. However, a member of the team periodically assisted or observed them.
Materials	Formalin Optimal Cutting Temperature (OCT) Cryomold Blade (VWR Cat #: 95057-832, coated microtone blades) Cryostat Staining Glass Container and Acetone 2% BSA+PBS Fluorescent mounting medium (Dako, S3023) Conjugated Antibody: Biolegend FITC anti-mouse Ly6G (clone 1A8), cat#:127606. BD Pharmingen PE anti-mouse CD49b (clone HMalpha), cat#:558759. eBioscience APC anti-mouse CD45 (clone 30-F11), cat#:17-0451-83. Fluorescent Microscope
Protocol	Tissue samples were collected from the lumbar region of the mice after blood draw and euthanization. Tissue Fixation Formalin: Remove the wax that from the application of Nair during the application of the transdermal patch. Treat tissue with the formalin solution to fix it the skin tissue samples. Store fixed skin tissue samples -80°C. Cryosection Fill an ice box with dry ice, ethanol, and a piece of metal. Place Optimal Cutting Temperature (OCT) compound and fixed tissue in a cryomold. Ensure the orientation of the tissue is such that all the layers of the tissue are obtained. Fill cryomold with OCT compound to cover the fixed tissue (approximately 0.5 cm from top of the mold). When samples are completely frozen,wrap them in individually-labelled foils, seal in plastic bags, and store at -80°C. Put the blade (VWR Cat #: 95057-832, coated microtone blades) in the Cryostat machine and tighten. Anchor and adjust tissue sample so it is parallel to the blade. Prime the tissue sample and slowly start cutting . Allow the section(s) to dry for approximately 3 hrs. Discard the blade. Cryosection FIxation Insert tissue slide in staining glass container that containing Acetone. Incubate at room temperature for 10 minutes. Fill staining glass container with the slides with prepared 2% BSA+PBS. Incubate at room temperature for 2 hours. Wipe the slide in such a way that a dome shape is created, and added 2% BSA+PBS to the dome. Add 1 uL of conjugated antibody to the dome. Incubate at room temperature for 1 hr. Ensure to add water to keep the slide moist at all times. Antibodies used are listed below: Biolegend FITC anti-mouse Ly6G (clone 1A8), cat#:127606 BD Pharmingen PE anti-mouse CD49b (clone HMalpha), cat#:558759 eBioscience APC anti-mouse CD45 (clone 30-F11), cat#:17-0451-83 Wash the slide three times with 2% BSA+PBS. Add 1 drop of Fluorescent mounting medium (Dako, S3023) to slide. Gently load the cover slip. Keep the tissue slide at 4°C. Visualize sample via fluorescent microscopy.

References

Haling, S. (1997). Super-rich medium. Biochemistry(16), 2280-2884.

Löbrich, M., Shibata, A., Beucher, A., Fisher, A., Ensminger, M., Goodarzi, A. a., … Jeggo, P. a. (2010). H2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization. Cell Cycle, 9(4), 662–669.

Spizizen, J. (1958). Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proceedings of the National Academy of Sciences of the United States of America(44), 1072-1078.

Difference between revisions of "Team:UofC Calgary/Experiments"

Latest revision as of 01:05, 20 October 2016

Experiments

iGEM

Latest Entries

Fully Trained!

Latest Pictures

Find us

@@ Line 96: / Line 96: @@
 								</li>
 								<li>
-									<a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Basic Parts</a>
+									<a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Parts Registry</a>
   <li>
                                                                   <a href="https://2016.igem.org/Team:UofC_Calgary/Composite_Part">Composite Parts</a>
@@ Line 5,530: / Line 5,530: @@
                      </ol>
                      <ul>
-                         <li class="c0"><li class="c8"><span class="c3 c32">50 ng digested vector DNA</span>
+                         <li class="c0"><span class="c3 c32">50 ng digested vector DNA</span>
                          </li>
-                         <li class="c0"><li class="c8"><span class="c3 c32">appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector</span>
+                         <li class="c0"><span class="c3 c32">appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector</span>
                          </li>
-                         <li class="c0"><li class="c8"><span class="c3 c32">1 &mu;L T4 DNA ligase</span>
+                         <li class="c0"><span class="c3 c32">1 &mu;L T4 DNA ligase</span>
                          </li>
-                         <li class="c0"><li class="c8"><span class="c3 c32">2 &mu;L 10X T4 DNA ligase buffer</span>
+                         <li class="c0"><span class="c3 c32">2 &mu;L 10X T4 DNA ligase buffer</span>
                          </li>
-                         <li class="c0"><li class="c8"><span class="c3 c32">ddH<sub>2</sub>O to a total volume of 20 &mu;L</span>
+                         <li class="c0"><span class="c3 c32">ddH<sub>2</sub>O to a total volume of 20 &mu;L</span>
                          </li>
                      </ul>
@@ Line 5,673: / Line 5,673: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">Various antibiotics (kanamycin, chloramphenicol, ampicillin and hygromycin) were used to select for successful </span><span class="c3 c15">E. coli </span><span class="c3">and </span><span class="c3 c15">B. subtilis </span><span class="c9 c3">transformants.</span>
+                     <p class="c38 c34"><span class="c3 c32">Various antibiotics (kanamycin, chloramphenicol, ampicillin and hygromycin) were used to select for successful <i>E. coli</i> and <i>B. subtilis</i> transformants.</span>
                      </p>
                  </td>
@@ Line 5,683: / Line 5,683: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Luria-Bertani broth with agar</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth with agar</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">10% (w/v) tryptone</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">5% (w/v) NaCl</span>
+                         <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">10% (w/v) yeast extract</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">15% (w/v) agar</span>
+                         <li class="c0"><span class="c3 c32">15% (w/v) agar</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c9 c3">Appropriate antibiotic</span>
+                     <p class="c38 c34"><span class="c3 c32">Appropriate antibiotic</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c3">kanamycin (final concentration of 100 </span><span class="c5 c3">&mu;g/mL)</span>
+                         <li class="c0"><span class="c3 c32">kanamycin (final concentration of 100 &mu;g/mL)</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">chloramphenicol (final concentration of 30 &mu;g/mL)</span>
+                         <li class="c0"><span class="c3 c32">chloramphenicol (final concentration of 30 &mu;g/mL)</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">ampicillin (final concentration of 50 &mu;g/mL)</span>
+                         <li class="c0"><span class="c3 c32">ampicillin (final concentration of 50 &mu;g/mL)</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">hygromycin (final concentration of 100 &mu;g/mL)</span>
+                         <li class="c0"><span class="c3 c32">hygromycin (final concentration of 100 &mu;g/mL)</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c5 c3">1500 mL Erlenmeyer flask</span>
+                     <p class="c38 c34"><span class="c3 c32">1500 mL Erlenmeyer flask</span>
                      </p>
                  </td>
@@ Line 5,718: / Line 5,718: @@
                  <td class="c27" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_ugk2xbe5oobt-0 start" start="1">
-                         <li class="c0"><span class="c3">In a 1500 Erlenmeyer flask, add 10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar in 1000 mL dH</span><span class="c3 c7">2</span><span class="c9 c3">O. Dissolve solids and make sure to add a stir bar.</span>
+                         <li class="c8"><span class="c3 c32">In a 1500 Erlenmeyer flask, add 10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar in 1000 mL dH<sub>2</sub>O. Dissolve solids and make sure to add a stir bar.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Cover flask loosely with aluminum foil, secure with autoclave tape, and sterilize by autoclaving for 20 minutes.</span>
+                         <li class="c8"><span class="c3 c32">Cover flask loosely with aluminum foil, secure with autoclave tape, and sterilize by autoclaving for 20 minutes.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Remove agar from autoclave using oven mits. Allow agar to cool until warm to the touch before adding appropriate antibiotic. Stir on hot plate and magnetic stirrer for 30 seconds.</span>
+                         <li class="c8"><span class="c3 c32">Remove agar from autoclave using oven mitts. Allow agar to cool until warm to the touch before adding appropriate antibiotic. Stir on hot plate and magnetic stirrer for 30 seconds.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Pour agar into plates using aseptic technique.</span>
+                         <li class="c8"><span class="c3 c32">Pour agar into plates using aseptic technique.</span>
                          </li>
                      </ol>
@@ Line 5,751: / Line 5,751: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">Competent </span><span class="c3 c15">E. coli </span><span class="c3">cells were transformed with ligated plasmid to amplify them for further use in digest confirmation and </span><span class="c3 c15">B. subtilis </span><span class="c9 c3">transformation.</span>
+                     <p class="c38 c34"><span class="c3 c32">Competent <i>E. coli</i> cells were transformed with ligated plasmid to amplify them for further use in digest confirmation and <i>B. subtilis</i> transformation.</span>
                      </p>
                  </td>
@@ Line 5,761: / Line 5,761: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3 c15">Escherichia coli </span><span class="c9 c3">TOP10 cells</span>
+                     <p class="c38 c34"><span class="c3 c32"><i>Escherichia coli</i> TOP10 cells</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Luria-Bertani broth</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">10% (w/v) tryptone</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">5% (w/v) NaCl</span>
+                         <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">10% (w/v) yeast extract</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c9 c3">16x125 mm culture tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">250 mL Erlenmeyer flask</span>
+                     <p class="c38 c34"><span class="c3 c32">250 mL Erlenmeyer flask</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Spectrophotometer</span>
+                     <p class="c38 c34"><span class="c3 c32">Spectrophotometer</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Centrifuge</span>
+                     <p class="c38 c34"><span class="c3 c32">Centrifuge</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">50 mL Falcon tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mL Falcon tubes</span>
                      </p>
-                     <p class="c2"><span class="c3">50 mM CaCl</span><span class="c37 c26 c3 c19 c7">2</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mM CaCl</span><span class="c37 c26 c3 c19 c7">2</span>
                      </p>
-                     <p class="c2"><span class="c3">50 mM CaCl</span><span class="c3 c7">2</span><span class="c9 c3">, 15% glycerol</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mM CaCl</span><span class="c3 c7">2</span><span class="c9 c3">, 15% glycerol</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">1.5 mL microcentrifuge tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">1.5 mL microcentrifuge tubes</span>
                      </p>
                  </td>
@@ Line 5,798: / Line 5,798: @@
                  <td class="c17" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_yzt1xhfk9inp-0 start" start="1">
-                         <li class="c0"><span class="c3">Inoculate 5-10 mL of Luria-Bertani broth with </span><span class="c3 c15">E. coli </span><span class="c9 c3">TOP10 cells and allow it to incubate at 37&deg;C overnight, shaking at 200 rpm.</span>
+                         <li class="c8"><span class="c3 c32">Inoculate 5-10 mL of Luria-Bertani broth with <i>E. coli</i> TOP10 cells and allow it to incubate at 37&deg;C overnight, shaking at 200 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c3">Subculture 1 mL of </span><span class="c3 c15">E. coli </span><span class="c3">overnight culture in 49 mL fresh Luria-Bertani broth in a 250 Erlenmeyer flask. Incubate at 37&deg;C, shaking at 200 rpm until it reaches an OD</span><span class="c3 c7">600 </span><span class="c9 c3">of 0.4-0.6 (usually takes 2.5 hours).</span>
+                         <li class="c8"><span class="c3 c32">Subculture 1 mL of <i>E. coli</i> overnight culture in 49 mL fresh Luria-Bertani broth in a 250 Erlenmeyer flask. Incubate at 37&deg;C, shaking at 200 rpm until it reaches an OD<sub>600</sub> of 0.4-0.6 (usually takes 2.5 hours).</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Spin down cells in 50 mL Falcon tube at 8200 g for 10 minutes at 4 &deg;C.</span>
+                         <li class="c8"><span class="c3 c32">Spin down cells in 50 mL Falcon tube at 8200 g for 10 minutes at 4 &deg;C.</span>
                          </li>
-                         <li class="c0"><span class="c3">Resuspend cells in 12.5 mL cold 50 mM CaCl</span><span class="c3 c7">2</span><span class="c9 c3">&nbsp;and place on ice for 10 minutes.</span>
+                         <li class="c8"><span class="c3 c32">Resuspend cells in 12.5 mL cold 50 mM CaCl<sub>2</sub> and place on ice for 10 minutes.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat Step 3.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Step 3.</span>
                          </li>
-                         <li class="c0"><span class="c3">Resuspend cells in 2 mL cold 50 mM CaCl</span><span class="c3 c7">2</span><span class="c9 c3">, 15% glycerol and place on ice for 30 minutes.</span>
+                         <li class="c8"><span class="c3 c32">Resuspend cells in 2 mL cold 50 mM CaCl<sub>2</sub>, 15% glycerol and place on ice for 30 minutes.</span>
                          </li>
-                         <li class="c0"><span class="c3">Separate cells into aliquots of 200 </span><span class="c12 c3">&mu;L and store at -80&deg;C.</span>
+                         <li class="c8"><span class="c3 c32">Separate cells into aliquots of 200 &mu;L and store at -80&deg;C.</span>
                          </li>
                      </ol>
@@ Line 5,837: / Line 5,837: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">Glycerol stocks of transformed </span><span class="c3 c15">E. coli </span><span class="c3">and </span><span class="c3 c15">B. subtilis </span><span class="c3">were prepared for long-term storage and future use. </span>
+                     <p class="c38 c34"><span class="c3 c32">Glycerol stocks of transformed <i>E. coli</i> and <i>B. subtilis</i> were prepared for long-term storage and future use. </span>
                      </p>
                  </td>
@@ Line 5,847: / Line 5,847: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Overnight culture of transformed bacteria</span>
+                     <p class="c38 c34"><span class="c3 c32">Overnight culture of transformed bacteria</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Sterile 1.5 mL cryo-tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">Sterile 1.5 mL cryo-tubes</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Sterile 50% glycerol</span>
+                     <p class="c38 c34"><span class="c3 c32">Sterile 50% glycerol</span>
                      </p>
                  </td>
@@ Line 5,862: / Line 5,862: @@
                  <td class="c27" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_9z0i9ngly08f-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">Using aseptic technique, pipette 0.5 mL of 50% sterile glycerol into a 1.5 mL cryo-tube.</span>
+                         <li class="c8"><span class="c3 c32">Using aseptic technique, pipette 0.5 mL of 50% sterile glycerol into a 1.5 mL cryo-tube.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Using aseptic technique, add 0.5 mL of overnight culture.</span>
+                         <li class="c8"><span class="c3 c32">Using aseptic technique, add 0.5 mL of overnight culture.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Pipette up and down gently to mix.</span>
+                         <li class="c8"><span class="c3 c32">Pipette up and down gently to mix.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Store at -80&deg;C for up to 1 year.</span>
+                         <li class="c8"><span class="c3 c32">Store at -80&deg;C for up to 1 year.</span>
                          </li>
                      </ol>
@@ Line 5,895: / Line 5,895: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Culture broth was plated on agar to isolate single colonies.</span>
+                     <p class="c38 c34"><span class="c3 c32">Culture broth was plated on agar to isolate single colonies.</span>
                      </p>
                  </td>
@@ Line 5,905: / Line 5,905: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Luria-Bertani agar plate with appropriate antibiotic (if required)</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani agar plate with appropriate antibiotic (if required)</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Overnight culture of desired bacteria</span>
+                     <p class="c38 c34"><span class="c3 c32">Overnight culture of desired bacteria</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">70% ethanol</span>
+                     <p class="c38 c34"><span class="c3 c32">70% ethanol</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Spreading rod</span>
+                     <p class="c38 c34"><span class="c3 c32">Spreading rod</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Bunsen burner</span>
+                     <p class="c38 c34"><span class="c3 c32">Bunsen burner</span>
                      </p>
                  </td>
@@ Line 5,924: / Line 5,924: @@
                  <td class="c17" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_t27wwiwmqyyi-0 start" start="1">
-                         <li class="c0"><span class="c3">Using aseptic technique, pipette 50-100 </span><span class="c5 c3">&mu;L of bacterial culture onto antibiotic agar plate.</span>
+                         <li class="c8"><span class="c3 c32">Using aseptic technique, pipette 50-100 &mu;L of bacterial culture onto antibiotic agar plate.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Dip spreading rod in 70% ethanols, pass over flame and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture.</span>
+                         <li class="c8"><span class="c3 c32">Dip spreading rod in 70% ethanols, pass over flame and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Use rod to spread bacterial culture over entire plate, spinning the plate at the same time.</span>
+                         <li class="c8"><span class="c3 c32">Use rod to spread bacterial culture over entire plate, spinning the plate at the same time.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Dip spreading rod in 70% ethanol, pass over flame and allow for excess liquid to burn off.</span>
+                         <li class="c8"><span class="c3 c32">Dip spreading rod in 70% ethanol, pass over flame and allow for excess liquid to burn off.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Incubate plates at 37&deg;C overnight or until growth is observed.</span>
+                         <li class="c8"><span class="c3 c32">Incubate plates at 37&deg;C overnight or until growth is observed.</span>
                          </li>
                      </ol>
@@ Line 5,959: / Line 5,959: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Culture broth was streaked on agar plates to isolate single colonies.</span>
+                     <p class="c38 c34"><span class="c3 c32">Culture broth was streaked on agar plates to isolate single colonies.</span>
                      </p>
                  </td>
@@ Line 5,969: / Line 5,969: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Luria-Bertani agar plate with appropriate antibiotic (if required)</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani agar plate with appropriate antibiotic (if required)</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Overnight culture of desired bacteria or single isolated colony on agar plate</span>
+                     <p class="c38 c34"><span class="c3 c32">Overnight culture of desired bacteria or single isolated colony on agar plate</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Inoculation loop</span>
+                     <p class="c38 c34"><span class="c3 c32">Inoculation loop</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Bunsen burner</span>
+                     <p class="c38 c34"><span class="c3 c32">Bunsen burner</span>
                      </p>
                  </td>
@@ Line 5,986: / Line 5,986: @@
                  <td class="c27" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_pgbvm1degh2i-0 start" start="1">
-                         <li class="c0"><span class="c3">Using aseptic technique, flame inoculation loop until red hot</span><span class="c5 c3">. Allow it to cool for 10 seconds or touch it to agar.</span>
+                         <li class="c8"><span class="c3 c32">Using aseptic technique, flame inoculation loop until red hot</span><span class="c5 c3">. Allow it to cool for 10 seconds or touch it to agar.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Dip the inoculation loop in bacterial culture or touch a single colony and streak the loop on &frac14; of the surface of agar in a zigzag motion.</span>
+                         <li class="c8"><span class="c3 c32">Dip the inoculation loop in bacterial culture or touch a single colony and streak the loop on &frac14; of the surface of agar in a zigzag motion.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.</span>
+                         <li class="c8"><span class="c3 c32">Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1.4 of the surface of the agar.</span>
+                         <li class="c8"><span class="c3 c32">Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1.4 of the surface of the agar.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Repeat Steps 3 and 4 two more times.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Steps 3 and 4 two more times.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Flame the inoculation loop until red hot. Allow it to cool for 10 seconds.</span>
+                         <li class="c8"><span class="c3 c32">Flame the inoculation loop until red hot. Allow it to cool for 10 seconds.</span>
                          </li>
-                         <li class="c0"><span class="c12 c3">Incubate plates at 37&deg;C overnight or until growth is observed.</span>
+                         <li class="c8"><span class="c3 c32">Incubate plates at 37&deg;C overnight or until growth is observed.</span>
                          </li>
                      </ol>
@@ Line 6,025: / Line 6,025: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Digested and undigested plasmids were run on agarose gels to confirm the presence and proper orientation of DNA inserts.</span>
+                     <p class="c38 c34"><span class="c3 c32">Digested and undigested plasmids were run on agarose gels to confirm the presence and proper orientation of DNA inserts.</span>
                      </p>
                  </td>
@@ Line 6,035: / Line 6,035: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">TAE buffer</span>
+                     <p class="c38 c34"><span class="c3 c32">TAE buffer</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">40 mM Tris, pH 7.6</span>
+                         <li class="c0"><span class="c3 c32">40 mM Tris, pH 7.6</span>
                          </li>
-                         <li class="c0"><span class="c3">20 mM CH</span><span class="c3 c7">3</span><span class="c9 c3">COOH</span>
+                         <li class="c0"><span class="c3 c32">20 mM CH<sub>3</sub>COOH</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">1 mM EDTA</span>
+                         <li class="c0"><span class="c3 c32">1 mM EDTA</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c9 c3">Agarose</span>
+                     <p class="c38 c34"><span class="c3 c32">Agarose</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">250 mL Erlenmeyer flask</span>
+                     <p class="c38 c34"><span class="c3 c32">250 mL Erlenmeyer flask</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">RedSafe Nucleic Acid Staining Solution</span>
+                     <p class="c38 c34"><span class="c3 c32">RedSafe Nucleic Acid Staining Solution</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Gel casting tray and comb</span>
+                     <p class="c38 c34"><span class="c3 c32">Gel casting tray and comb</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">10X loading dye</span>
+                     <p class="c38 c34"><span class="c3 c32">10X loading dye</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">DNA sample</span>
+                     <p class="c38 c34"><span class="c3 c32">DNA sample</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Microwave</span>
+                     <p class="c38 c34"><span class="c3 c32">Microwave</span>
                      </p>
                  </td>
@@ Line 6,068: / Line 6,068: @@
                  <td class="c17" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_jwmzf1bv57q2-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">For a 1% gel (standard), add 1 g agarose to 100 mL TAE buffer in a 250 mL Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long).</span>
+                         <li class="c8"><span class="c3 c32">For a 1% gel (standard), add 1 g agarose to 100 mL TAE buffer in a 250 mL Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long).</span>
                          </li>
-                         <li class="c0"><span class="c3">Allow flask to cool in fumehood until warm to the touch before adding 5 </span><span class="c5 c3">&mu;L RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.</span>
+                         <li class="c8"><span class="c3 c32">Allow flask to cool in fumehood until warm to the touch before adding 5 &mu;L RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.</span>
+                         <li class="c8"><span class="c3 c32">Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.</span>
+                         <li class="c8"><span class="c3 c32">Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Load samples of 20 &mu;L DNA containing 2 &mu;L loading dye.</span>
+                         <li class="c8"><span class="c3 c32">Load samples of 20 &mu;L DNA containing 2 &mu;L loading dye.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Run gel at 120 V for 45 minutes or until loading dye is &#8532; way down the gel.</span>
+                         <li class="c8"><span class="c3 c32">Run gel at 120 V for 45 minutes or until loading dye is &#8532; way down the gel.</span>
                          </li>
                      </ol>
@@ Line 6,105: / Line 6,105: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">To simulate the temperature conditions of </span><span class="c3 c15">B. subtilis </span><span class="c3">in our patch, we measured bacterial growth in 10 mL of Luria-Bertani (LB) broth over the course of 24 hours at varying temperatures of 35&deg;C (average skin temperature), 22&deg;C (average room temperature on board the International Space Station) and 4&deg;C (negative control). Growth was measured by spectrophotometer at a wavelength of 600 nm (OD</span><span class="c7">600</span><span class="c9 c3">).</span>
+                     <p class="c38 c34"><span class="c3 c32">To simulate the temperature conditions of <i>B. subtilis</i> in our patch, we measured bacterial growth in 10 mL of Luria-Bertani (LB) broth over the course of 24 hours at varying temperatures of 35&deg;C (average skin temperature), 22&deg;C (average room temperature on board the International Space Station) and 4&deg;C (negative control). Growth was measured by spectrophotometer at a wavelength of 600 nm (OD<sub>600</sub>).</span>
                      </p>
                  </td>
@@ Line 6,115: / Line 6,115: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Luria-Bertani broth medium with hygromycin</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth medium with hygromycin</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">10% (w/v) tryptone</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">5% (w/v) NaCl</span>
+                         <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">10% (w/v) yeast extract</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span>
                          </li>
-                         <li class="c0"><span class="c3">Final concentration of 100 </span><span class="c12 c3">&mu;g/mL hygromycin</span>
+                         <li class="c0"><span class="c3 c32">Final concentration of 100 &mu;g/mL hygromycin</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c3 c15">Bacillus subtilis </span><span class="c9 c3">WB800 cells</span>
+                     <p class="c38 c34"><span class="c3 c32"><i>Bacillus subtilis</i> WB800 cells</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">16x125 mm culture tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">50 mL Falcon tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mL Falcon tubes</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Spectrophotometer</span>
+                     <p class="c38 c34"><span class="c3 c32">Spectrophotometer</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">1 mL cuvette</span>
+                     <p class="c38 c34"><span class="c3 c32">1 mL cuvette</span>
                      </p>
                  </td>
@@ Line 6,145: / Line 6,145: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c4 c3 c30">Preparation of </span><span class="c4 c3 c30 c15">B. subtilis </span><span class="c13 c4 c3">WB800 Cells</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Preparation of <i>B. subtilis</i> WB800 Cells</b></span>
                      </p>
                      <ol class="c10 lst-kix_e6kd68i2bo5-0 start" start="1">
-                         <li class="c0"><span class="c3">Two days prior to measuring growth, prepare an overnight culture of </span><span class="c3 c15">B. subtilis </span><span class="c3">in 10 mL LB broth with the appropriate antibiotic (100 </span><span class="c12 c3">&mu;</span><span class="c9 c3">g/mL final concentration of hygromycin). Incubate at 37&deg;C, shaking at 200 rpm overnight.</span>
+                         <li class="c8"><span class="c3 c32">Two days prior to measuring growth, prepare an overnight culture of <i>B. subtilis</i> in 10 mL LB broth with the appropriate antibiotic (100 &mu;g/mL final concentration of hygromycin). Incubate at 37&deg;C, shaking at 200 rpm overnight.</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Inoculation</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Inoculation</b></span>
                      </p>
                      <ol class="c10 lst-kix_e6kd68i2bo5-0" start="2">
-                         <li class="c0"><span class="c9 c3">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35&deg;C, 22&deg;C or 4&deg;C) with lids loosened, shaking at 200 rpm.</span>
+                         <li class="c8"><span class="c3 c32">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35&deg;C, 22&deg;C or 4&deg;C) with lids loosened, shaking at 200 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c4 c3 c13">Measurement of Growth</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Measurement of Growth</b></span>
                      </p>
                      <ol class="c10 lst-kix_e6kd68i2bo5-0" start="5">
-                         <li class="c0"><span class="c3">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD</span><span class="c7">600</span><span class="c3">). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, </span><span class="c24 c26 c4 c3">making sure to only blank once!</span>
+                         <li class="c8"><span class="c3 c32">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD<sub>600</sub>). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, <b>making sure to only blank once!</b></span>
                          </li>
-                         <li class="c0"><span class="c3">Commence measurement of samples at 8:00 (OD</span><span class="c7">600</span><span class="c3">). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span>
+                         <li class="c8"><span class="c3 c32">Commence measurement of samples at 8:00 (OD<sub>600</sub>). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span>
                          </li>
-                         <li class="c0"><span class="c12 c3">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. </span><span class="c3">&nbsp;If samples reach an OD</span><span class="c7">600</span><span class="c3">&nbsp;&gt; 0.4, dilute the sample 10X using LB-broth with 100 </span><span class="c12 c3">&mu;g/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.</span>
+                         <li class="c8"><span class="c3 c32">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD<sub>600</sub> >0.4, dilute the sample 10X using LB-broth with 100 &mu;g/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.</span>
                          </li>
                      </ol>
@@ Line 6,195: / Line 6,195: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">Our patch will contain three attached packets of additional media that can be ruptured to extend the cell growth and production of mBBI. In order to determine which media would be best suited for these packets, we tested three different types of media: Luria-Bertani broth, 2X Luria-Bertani broth, and Super Rich broth. We added 1 mL of these three types of media to 9 mL cultures of </span><span class="c3 c15">B. subtilis </span><span class="c3">WB800 in LB broth containing 100 </span><span class="c12 c3">&mu;g/mL hygromycin every 12 hours and measured bacterial growth over the course of 72 hours. </span><span class="c3">Growth was measured by spectrophotometer at a wavelength of 600 nm (OD</span><span class="c7">600</span><span class="c9 c3">).</span>
+                     <p class="c38 c34"><span class="c3 c32">Our patch will contain three attached packets of additional media that can be ruptured to extend the cell growth and production of mBBI. In order to determine which media would be best suited for these packets, we tested three different types of media: Luria-Bertani broth, 2X Luria-Bertani broth, and Super Rich broth. We added 1 mL of these three types of media to 9 mL cultures of <i>B. subtilis</i> WB800 in LB broth containing 100 &mu;g/mL hygromycin every 12 hours and measured bacterial growth over the course of 72 hours. Growth was measured by spectrophotometer at a wavelength of 600 nm (OD<sub>600</sub>).</span>
                      </p>
                  </td>
@@ Line 6,205: / Line 6,205: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Luria-Bertani broth medium with hygromycin</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth medium with hygromycin</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">10% (w/v) tryptone</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">5% (w/v) NaCl</span>
+                         <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">10% (w/v) yeast extract</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span>
                          </li>
-                         <li class="c0"><span class="c3">Final concentration of 100 </span><span class="c5 c3">&mu;g/mL hygromycin</span>
+                         <li class="c0"><span class="c3 c32">Final concentration of 100 &mu;g/mL hygromycin</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c5 c3">2X Luria-Bertani broth medium with hygromycin</span>
+                     <p class="c38 c34"><span class="c3 c32">2X Luria-Bertani broth medium with hygromycin</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">20% (w/v) tryptone</span>
+                         <li class="c0"><span class="c3 c32">20% (w/v) tryptone</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">10% (w/v) NaCl</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) NaCl</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">20% (w/v) yeast extract</span>
+                         <li class="c0"><span class="c3 c32">20% (w/v) yeast extract</span>
                          </li>
-                         <li class="c0"><span class="c3">Final concentration of 100 </span><span class="c5 c3">&mu;g/mL hygromycin</span>
+                         <li class="c0"><span class="c3 c32">Final concentration of 100 &mu;g/mL hygromycin</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c5 c3">Super Rich broth medium</span>
+                     <p class="c38 c34"><span class="c3 c32">Super Rich broth medium</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c5 c3">2.5% (w/v) tryptose</span>
+                         <li class="c0"><span class="c3 c32">2.5% (w/v) tryptose</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">2% (w/v) yeast extract</span>
+                         <li class="c0"><span class="c3 c32">2% (w/v) yeast extract</span>
                          </li>
-                         <li class="c0"><span class="c12 c3">0.3% K</span><span class="c12 c3 c7">2</span><span class="c12 c3">HPO</span><span class="c12 c26 c3 c19 c7 c46">4</span>
+                         <li class="c0"><span class="c3 c32">0.3% K<sub>2</sub>HPO<sub>4</sub>
                          </li>
-                         <li class="c0"><span class="c5 c3">3% glucose (v/v)</span>
+                         <li class="c0"><span class="c3 c32">3% glucose (v/v)</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Notes:</span>
+                         <li class="c0"><span class="c3 c32">Notes:</span>
                          </li>
                      </ul>
                      <ul>
-                         <li class="c2 c39"><span class="c5 c3">Mix all components except glucose and adjust pH to 7.5</span>
+                         <li class="c2 c39"><span class="c3 c32">Mix all components except glucose and adjust pH to 7.5</span>
                          </li>
-                         <li class="c2 c39"><span class="c5 c3">Autoclave solution except for glucose. Add 1 mL/L of anti-foam prior to autoclave.</span>
+                         <li class="c2 c39"><span class="c3 c32">Autoclave solution except for glucose. Add 1 mL/L of anti-foam prior to autoclave.</span>
                          </li>
-                         <li class="c2 c39"><span class="c5 c3">Filter-sterilize glucose (20%) and add to main solution to a final concentration of 3%.</span>
+                         <li class="c2 c39"><span class="c3 c32">Filter-sterilize glucose (20%) and add to main solution to a final concentration of 3%.</span>
                          </li>
-                         <li class="c2 c39"><span class="c12 c3">Adapted from </span><span class="c3">Haling </span><span class="c3 c15">et al.</span><span class="c3">, 1997</span>
+                         <li class="c2 c39"><span class="c3 c32">Adapted from Haling <i>et al.,</i> 1997</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c3 c15">Bacillus subtilis </span><span class="c9 c3">WB800 cells</span>
+                     <p class="c38 c34"><span class="c3 c32"><i>Bacillus subtilis</i> WB800 cells</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">16x125 mm culture tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">50 mL Falcon tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mL Falcon tubes</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Spectrophotometer</span>
+                     <p class="c38 c34"><span class="c3 c32">Spectrophotometer</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">1 mL cuvette</span>
+                     <p class="c38 c34"><span class="c3 c32">1 mL cuvette</span>
                      </p>
                  </td>
@@ Line 6,271: / Line 6,271: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c4 c3 c30">Preparation of </span><span class="c4 c3 c30 c15">B. subtilis </span><span class="c13 c4 c3">WB800 Cells</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Preparation of <i>B. subtilis</i> WB800 Cells</b></span>
                      </p>
                      <ol class="c10 lst-kix_l5833w3zpti7-0 start" start="1">
-                        <li class="c0"><span class="c3">Two days prior to measuring growth, prepare an overnight culture of </span><span class="c3 c15">B. subtilis </span><span class="c3">in 10 mL LB broth with the appropriate antibiotic (100 </span><span class="c12 c3">&mu;</span><span class="c9 c3">g/mL final concentration of hygromycin). Incubate at 35&deg;C, shaking at 200 rpm overnight.</span>
+                       <li class="c8"><span class="c3 c32">Two days prior to measuring growth, prepare an overnight culture of <i>B. subtilis</i> in 10 mL LB broth with the appropriate antibiotic (100 &mu;g/mL final concentration of hygromycin). Incubate at 35&deg;C, shaking at 200 rpm overnight.</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Inoculation</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Inoculation</b></span>
                      </p>
                      <ol class="c10 lst-kix_l5833w3zpti7-0" start="2">
-                         <li class="c0"><span class="c9 c3">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at 37&deg;C with lids loosened, shaking at 200 rpm.</span>
+                         <li class="c8"><span class="c3 c32">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at 37&deg;C with lids loosened, shaking at 200 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Measurement of Growth</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Measurement of Growth</b></span>
                      </p>
                      <ol class="c10 lst-kix_l5833w3zpti7-0" start="5">
-                         <li class="c0"><span class="c3">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD</span><span class="c7">600</span><span class="c3">). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, </span><span class="c24 c4 c3 c26">making sure to only blank once!</span>
+                         <li class="c8"><span class="c3 c32">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD<sub>600</sub>). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once!</span>
                          </li>
-                         <li class="c0"><span class="c3">Commence measurement of samples at 8:00 (OD</span><span class="c7">600</span><span class="c3">). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span>
+                         <li class="c8"><span class="c3 c32">Commence measurement of samples at 8:00 (OD<sub>600</sub>). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span>
                          </li>
-                         <li class="c0"><span class="c12 c3">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. </span><span class="c3">&nbsp;If samples reach an OD</span><span class="c7">600</span><span class="c3">&nbsp;&gt; 0.4, dilute the sample 10X using LB-broth with 100 </span><span class="c5 c3">&mu;g/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to incubate at 35&deg;C with lids loosened, shaking at 200 rpm.</span>
+                         <li class="c8"><span class="c3 c32">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD<sub>600</sub> >0.4, dilute the sample 10X using LB-broth with 100 &mu;g/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to incubate at 35&deg;C with lids loosened, shaking at 200 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Add 1 mL of appropriate media (either LB, 2XLB or Super Rich) to each 50 mL Falcon tube every 12 hours for a total of three times (at 12 hours, 24 hours and 36 hours) after inoculation.</span>
+                         <li class="c8"><span class="c3 c32">Add 1 mL of appropriate media (either LB, 2XLB or Super Rich) to each 50 mL Falcon tube every 12 hours for a total of three times (at 12 hours, 24 hours and 36 hours) after inoculation.</span>
                          </li>
                      </ol>
@@ Line 6,323: / Line 6,323: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3 c15">Escherichia coli </span><span class="c3">TOP10 cells were used to amplify newly-ligated plasmids and were later harvested for future use in digest confirmation and </span><span class="c3 c15">B. subtilis </span><span class="c9 c3">transformation.</span>
+                     <p class="c38 c34"><span class="c3 c32"><i>Escherichia coli</i> TOP10 cells were used to amplify newly-ligated plasmids and were later harvested for future use in digest confirmation and <i>Bacillus subtilis</i> transformation.</span>
                      </p>
                  </td>
@@ Line 6,333: / Line 6,333: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">Competent </span><span class="c3 c15">E. coli </span><span class="c3">TOP10 aliquots (200 </span><span class="c5 c3">&mu;L)</span>
+                     <p class="c38 c34"><span class="c3 c32">Competent <i>E. coli</i> TOP10 aliquots (200 &mu;L)</span>
                      </p>
-                     <p class="c2"><span class="c5 c3">DNA for transformation</span>
+                     <p class="c38 c34"><span class="c3 c32">DNA for transformation</span>
                      </p>
-                     <p class="c2"><span class="c5 c3">Luria-Bertani broth</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">10% (w/v) tryptone</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">5% (w/v) NaCl</span>
+                         <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span>
                          </li>
-                         <li class="c0"><span class="c3">10% (w/v) yeast extract</span>
+                         <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c5 c3">Agar plate with appropriate antibiotic</span>
+                     <p class="c38 c34"><span class="c3 c32">Agar plate with appropriate antibiotic</span>
                      </p>
                  </td>
@@ Line 6,358: / Line 6,358: @@
                  <td class="c17" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_3v73ll15y5y-0 start" start="1">
-                         <li class="c0"><span class="c3">Thaw 200 </span><span class="c12 c3">&mu;L aliquot of competent </span><span class="c12 c3 c15">E. coli</span><span class="c5 c3">&nbsp;TOP10 cells on ice just before use.</span>
+                         <li class="c8"><span class="c3 c32">Thaw 200 &mu;L aliquot of competent <i>E. coli</i> TOP10 cells on ice just before use.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Add 0.3-1 &mu;g DNA to cells (in maximum 20 &mu;L), flick gently to mix, and place on ice for 30 minutes.</span>
+                         <li class="c8"><span class="c3 c32">Add 0.3-1 &mu;g DNA to cells (in maximum 20 &mu;L), flick gently to mix, and place on ice for 30 minutes.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Heat shock for 60-75 seconds at 42&deg;C.</span>
+                         <li class="c8"><span class="c3 c32">Heat shock for 60-75 seconds at 42&deg;C.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Place on ice for 5 minutes.</span>
+                         <li class="c8"><span class="c3 c32">Place on ice for 5 minutes.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Add 250 &mu;L Luria-Bertani medium to aliquot of cells.</span>
+                         <li class="c8"><span class="c3 c32">Add 250 &mu;L Luria-Bertani medium to aliquot of cells.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Incubate cells for 60 minutes at 37&deg;C, shaking at 200 rpm for 1 hour.</span>
+                         <li class="c8"><span class="c3 c32">Incubate cells for 60 minutes at 37&deg;C, shaking at 200 rpm for 1 hour.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Pellet cells in a microcentrifuge at 3500 g for 1 min and discard supernatant.</span>
+                         <li class="c8"><span class="c3 c32">Pellet cells in a microcentrifuge at 3500 g for 1 min and discard supernatant.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Resuspend pellet in 250 &mu;L Luria-Bertani broth.</span>
+                         <li class="c8"><span class="c3 c32">Resuspend pellet in 250 &mu;L Luria-Bertani broth.</span>
                          </li>
-                         <li class="c0"><span class="c3 c5">Plate 50-100 &mu;L of resuspended culture on agar plate with appropriate antibiotic and spread.</span>
+                         <li class="c8"><span class="c3 c32">Plate 50-100 &mu;L of resuspended culture on agar plate with appropriate antibiotic and spread.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Incubate plates at 37&deg;C overnight or until desired growth is observed.</span>
+                         <li class="c8"><span class="c3 c32">Incubate plates at 37&deg;C overnight or until desired growth is observed.</span>
                          </li>
                      </ol>
@@ Line 6,403: / Line 6,403: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3 c15">Bacillus subtilis </span><span class="c9 c3">was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.</span>
+                     <p class="c38 c34"><span class="c3 c32"><i>Bacillus subtilis</i> was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.</span>
                      </p>
                  </td>
@@ Line 6,413: / Line 6,413: @@
                  </td>
                  <td class="c27" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">SP1 Salts</span>
+                     <p class="c38 c34"><span class="c3 c32">SP1 Salts</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c3">0.2% (w/v) (NH</span><span class="c3 c7">4</span><span class="c3">)</span><span class="c3 c7">2</span><span class="c3">SO</span><span class="c26 c3 c19 c7 c37">4</span>
+                         <li class="c0"><span class="c3 c32">>0.2% (w/v) (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub></span>
                          </li>
-                         <li class="c0"><span class="c3">1.4% (w/v) K</span><span class="c3 c7">2</span><span class="c3">HPO</span><span class="c3 c7">4</span>
+                         <li class="c0"><span class="c3 c32">1.4% (w/v) K<sub>2</sub>HPO<sub>4</sub></span>
                          </li>
-                         <li class="c0"><span class="c3">0.6% (w/v) KH</span><span class="c3 c7">2</span><span class="c3">PO</span><span class="c37 c26 c3 c19 c7">4</span>
+                         <li class="c0"><span class="c3 c32">0.6% (w/v) KH<sub>2</sub>PO<sub>4</sub></span>
                          </li>
-                         <li class="c0"><span class="c3">0.1% Na</span><span class="c3 c7">3</span><span class="c3">C</span><span class="c3 c7">6</span><span class="c3">H</span><span class="c3 c7">5</span><span class="c3">O</span><span class="c3 c7">7</span><span class="c12 c44">&sdot;</span><span class="c3">2H</span><span class="c3 c7">2</span><span class="c9 c3">O</span>
+                         <li class="c0"><span class="c3 c32">0.1% Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>O<sub>7</sub>&sdot;2H<sub>2</sub>O</span>
                          </li>
-                         <li class="c0"><span class="c3">0.02% MgSO</span><span class="c3 c7">4</span><span class="c12 c44">&sdot;</span><span class="c3">7H</span><span class="c3 c7">2</span><span class="c9 c3">O</span>
+                         <li class="c0"><span class="c3 c32">0.02% MgSO<sub>4</sub>&sdot;7H<sub>2</sub>O</span>
                          </li>
                      </ul>
-                     <p class="c2"><span class="c3">50 mM CaCl</span><span class="c37 c26 c3 c19 c7">2</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mM CaCl<sub>2</sub></span>
                      </p>
-                     <p class="c2"><span class="c3">250 mM MgCl</span><span class="c37 c26 c3 c19 c7">2</span>
+                     <p class="c38 c34"><span class="c3 c32">250 mM MgCl<sub>2</sub></span>
                      </p>
-                     <p class="c2"><span class="c9 c3">50% filter-sterilized glucose</span>
+                     <p class="c38 c34"><span class="c3 c32">50% filter-sterilized glucose</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Casamino acids/yeast extract</span>
+                     <p class="c38 c34"><span class="c3 c32">Casamino acids/yeast extract</span>
                      </p>
                      <ul>
-                         <li class="c0"><span class="c9 c3">2% (w/v) casamino acids</span>
+                         <li class="c0"><span class="c3 c32">2% (w/v) casamino acids</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">10% yeast extract</span>
+                         <li class="c0"><span class="c3 c32">10% yeast extract</span>
                          </li>
                      </ul>
@@ Line 6,450: / Line 6,450: @@
                  <td class="c27" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_pt735i4wbfer-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">For all stock solutions, autoclave solutions for 20 minutes and store at room temperature.</span>
+                         <li class="c8"><span class="c3 c32">For all stock solutions, autoclave solutions for 20 minutes and store at room temperature.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">To make SP1 media, add any volume of SP1 salts plus 1/100 volume glucose, 1/100 volume casamino acids/yeast extract, and 0.5% (w/v) tryptophan.</span>
+                         <li class="c8"><span class="c3 c32">To make SP1 media, add any volume of SP1 salts plus 1/100 volume glucose, 1/100 volume casamino acids/yeast extract, and 0.5% (w/v) tryptophan.</span>
                          </li>
-                         <li class="c0"><span class="c3">To make SP2 media, add 1/100 volume of CaCl</span><span class="c3 c7">2 </span><span class="c3">and 1/100 volume of MgCl</span><span class="c3 c7">2</span><span class="c9 c3">&nbsp;to any volume of SP1 media.</span>
+                         <li class="c8"><span class="c3 c32">To make SP2 media, add 1/100 volume of CaCl<sub>2</sub> and 1/100 volume of MgCl<sub>2</sub> to any volume of SP1 media.</span>
                          </li>
                      </ol>
@@ Line 6,481: / Line 6,481: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3 c15">B. subtilis </span><span class="c9 c3">was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.</span>
+                     <p class="c38 c34"><span class="c3 c32"><i>B. subtilis</i> was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.</span>
                      </p>
                  </td>
@@ Line 6,491: / Line 6,491: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Luria-Bertani agar plate with appropriate antibiotic</span>
+                     <p class="c38 c34"><span class="c3 c32">Luria-Bertani agar plate with appropriate antibiotic</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">16x125 mm culture tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">13x100 mm culture tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">13x100 mm culture tubes</span>
                      </p>
-                     <p class="c2"><span class="c3">Sterile SP1 Medium</span>
+                     <p class="c38 c34"><span class="c3 c32">Sterile SP1 Medium</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Sterile SP2 Medium</span>
+                     <p class="c38 c34"><span class="c3 c32">Sterile SP2 Medium</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">100 mM EGTA</span>
+                     <p class="c38 c34"><span class="c3 c32">100 mM EGTA</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">DNA for transformation</span>
+                     <p class="c38 c34"><span class="c3 c32">DNA for transformation</span>
                      </p>
                  </td>
@@ Line 6,514: / Line 6,514: @@
                  <td class="c17" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_pt735i4wbfer-0" start="4">
-                         <li class="c0"><span class="c9 c3">Streak cells on Luria-Bertani agar plate in the evening at 17:00. Incubate cells at 30&deg;C overnight.</span>
+                         <li class="c8"><span class="c3 c32">Streak cells on Luria-Bertani agar plate in the evening at 17:00. Incubate cells at 30&deg;C overnight.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">At 9:00 the next morning, transfer cells to 2 mL SP1 medium in 16x125 mm culture tubes. The culture should be slightly turbid. Incubate at 37&deg;C for 3 hours and 45 minutes, shaking at 300 rpm.</span>
+                         <li class="c8"><span class="c3 c32">At 9:00 the next morning, transfer cells to 2 mL SP1 medium in 16x125 mm culture tubes. The culture should be slightly turbid. Incubate at 37&deg;C for 3 hours and 45 minutes, shaking at 300 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Transfer 0.5 mL SP1 culture to 4.5 mL SP2 medium prewarmed to 37&deg;C in a 16x125 mm culture tube. Incubate at 37&deg;C for 1 hour and 30 minutes, shaking at 150 rpm.</span>
+                         <li class="c8"><span class="c3 c32">Transfer 0.5 mL SP1 culture to 4.5 mL SP2 medium pre-warmed to 37&deg;C in a 16x125 mm culture tube. Incubate at 37&deg;C for 1 hour and 30 minutes, shaking at 150 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c3">Add 50 </span><span class="c5 c3">&mu;L 100X EGTA to the SP2 culture and incubate at 37&deg;C for another 10 minutes, shaking at 150 rpm.</span>
+                         <li class="c8"><span class="c3 c32">Add 50 &mu;L 100X EGTA to the SP2 culture and incubate at 37&deg;C for another 10 minutes, shaking at 150 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Take 0.5 mL competent cells and transfer to a sterile 13x100 mm culture tube. Add 0.1-3 g DNA in a volume of 60 &mu;L or less. Incubate at 37&deg;C for 1 hour and 30 minutes, shaking at 300 rpm.</span>
+                         <li class="c8"><span class="c3 c32">Take 0.5 mL competent cells and transfer to a sterile 13x100 mm culture tube. Add 0.1-3 g DNA in a volume of 60 &mu;L or less. Incubate at 37&deg;C for 1 hour and 30 minutes, shaking at 300 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Plate 0.25 mL of sample on Luria-Bertani agar plate with appropriate antibiotic.</span>
+                         <li class="c8"><span class="c3 c32">Plate 0.25 mL of sample on Luria-Bertani agar plate with appropriate antibiotic.</span>
                          </li>
-                         <li class="c0"><span class="c5 c3">Incubate plate at 37&deg;C overnight or until desired growth is observed.</span>
+                         <li class="c8"><span class="c3 c32">Incubate plate at 37&deg;C overnight or until desired growth is observed.</span>
                          </li>
                      </ol>
@@ Line 6,559: / Line 6,559: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c9 c3">The purpose of this assay was to determine quantitatively if the membrane in our transdermal patch can act as a filter and prevent bacteria cells from diffusing through the semipermeable membrane. </span>
+                     <p class="c38 c34"><span class="c3 c32">The purpose of this assay was to determine quantitatively if the membrane in our transdermal patch can act as a filter and prevent bacteria cells from diffusing through the semipermeable membrane. </span>
                      </p>
                  </td>
@@ Line 6,569: / Line 6,569: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c9 c3">3 50 mL falcon tube</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mL falcon tubes</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">3 15 mL falcon tube</span>
+                     <p class="c38 c34"><span class="c3 c32">15 mL falcon tubes</span>
                      </p>
-                     <p class="c6"><span class="c3 c9">Distilled/non distilled water</span>
+                     <p class="c38 c34"><span class="c3 c32">Distilled/non distilled water</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">Sodium chloride</span>
+                     <p class="c38 c34"><span class="c3 c32">NaCl</span>
                      </p>
-                     <p class="c6"><span class="c3">20 mL overnight culture (</span><span class="c3 c15">B. subtilis </span><span class="c3">strain</span><span class="c36 c26 c3 c19 c15">) </span>
+                     <p class="c38 c34"><span class="c3 c32">20 mL overnight culture (<i>B. subtilis </i> WB800) </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">LB media </span>
+                     <p class="c38 c34"><span class="c3 c32">LB media </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">CoTran 9716 and 9728 semipermeable membrane </span>
+                     <p class="c38 c34"><span class="c3 c32">CoTran 9716 and 9728 semipermeable membrane </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">Parafilm</span>
+                     <p class="c38 c34"><span class="c3 c32">Parafilm</span>
                      </p>
                  </td>
@@ Line 6,593: / Line 6,593: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c13 c4 c3">Preparation</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Preparation</b></span>
                      </p>
                      <ol class="c10 lst-kix_4jptj2dntjck-0 start" start="1">
-                         <li class="c6 c11"><span class="c9 c3">Prepare 500 mL of 0.9% W/V saline solution using sodium chloride at a pH of 7.35. Cover using parafilm and store at room temperature</span>
+                         <li class="c8"><span class="c3 c32">Prepare 500 mL of 0.9% w/v saline solution using sodium chloride at a pH of 7.35. Cover using parafilm and store at room temperature.</span>
                          </li>
-                         <li class="c6 c11"><span class="c3">Start 20 mL overnight culture </span><span class="c3 c15">B. subtilis</span>
+                         <li class="c8"><span class="c3 c32">Start 20 mL overnight culture <i>B. subtilis</i>.
                          </li>
-                         <li class="c6 c11"><span class="c3">For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of </span><span class="c3 c15">B. subtilis </span>
+                         <li class="c8"><span class="c3 c32">For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of <i>B. subtilis</i>.
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.</span>
+                         <li class="c8"><span class="c3 c32">Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Repeat process using cell culture in distilled water</span>
+                         <li class="c8"><span class="c3 c32">Repeat process using cell culture in distilled water.</span>
                          </li>
-                        <li class="c6 c11"><span class="c9 c3">Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay</span>
+                       <li class="c8"><span class="c3 c32">Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got</span>
+                         <li class="c8"><span class="c3 c32">As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got.</span>
                          </li>
                      </ol>
-                     <p class="c6"><span class="c13 c4 c3">Measurements</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Measurements</b></span>
                      </p>
                      <ol class="c10 lst-kix_pzhk3qcax242-0 start" start="1">
-                         <li class="c6 c11"><span class="c9 c3">Aliquot 5 mL of each overnight culture into three 15 mL falcon tubes</span>
+                         <li class="c8"><span class="c3 c32">Aliquot 5 mL of each overnight culture into three 15 mL falcon tubes.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">With one of the 15 mL falcon tube wrap parafilm over the top of the tube</span>
+                         <li class="c8"><span class="c3 c32">With one of the 15 mL falcon tube wrap parafilm over the top of the tube.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">With the other, place the 9716 semipermeable membrane over the top of the tube and parafilm around the edges to prevent it from falling off. Repeat using 9728 semipermeable membrane</span>
+                         <li class="c8"><span class="c3 c32">With the other, place the 9716 semipermeable membrane over the top of the tube and Parafilm around the edges to prevent it from falling off. Repeat using 9728 semipermeable membrane</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">With the 50 mL falcon tubes, add 30 mL of saline solution</span>
+                         <li class="c8"><span class="c3 c32">With the 50 mL falcon tubes, add 30 mL of saline solution</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Invert and submerse the 15 mL falcon tubes into the 50 mL falcon tubes. Cover the top of the 50 mL falcon tube with parafilm</span>
+                         <li class="c8"><span class="c3 c32">Invert and submerse the 15 mL falcon tubes into the 50 mL falcon tubes. Cover the top of the 50 mL falcon tube with Parafilm.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">After a 24 hour period take a 1 mL sample of the water in the 50 mL falcon tube </span>
+                         <li class="c8"><span class="c3 c32">After a 24 hour period take a 1 mL sample of the water in the 50 mL falcon tube.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm </span>
+                         <li class="c8"><span class="c3 c32">Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Repeat for seven days </span>
+                         <li class="c8"><span class="c3 c32">Repeat once daily for seven days.</span>
                          </li>
                      </ol>
@@ Line 6,655: / Line 6,655: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c9 c3">In running the first diffusion assay, our data analysis showed that there was an increased amount of cell detection, meaning that the cells may or may not be diffusing through. However, we suspected that there might be contamination so we repeated the assay with a few changes. </span>
+                     <p class="c38 c34"><span class="c3 c32">In running the first diffusion assay, our data analysis showed that there was an increased amount of cell detection, meaning that the cells may or may not be diffusing through. However, we suspected that there might be contamination so we repeated the assay with a few changes. </span>
                      </p>
                      <p class="c6 c28"><span class="c9 c3"></span>
                      </p>
-                     <p class="c6"><span class="c9 c3">This time, we are using a filter sterilization membrane that has pore sizes of 0.2 micron. We are also adding hygromycin to the saline solution for <i>B. subtilis</i> WB800 and chloramphenicol for E.coli GFP + mBBI. This ensured that there would be no contamination in our samples. &nbsp;</span>
+                     <p class="c38 c34"><span class="c3 c32">This time, we are using a filter sterilization membrane that has pore sizes of 0.2 micron. We are also adding hygromycin to the saline solution for <i>B. subtilis</i> WB800 and chloramphenicol for <i>E.coli</i> mBBI-GFP. This ensured that there would be no contamination in our samples. </span>
                      </p>
                  </td>
@@ Line 6,669: / Line 6,669: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c9 c3">3 &nbsp;10 mL syringes</span>
+                     <p class="c38 c34"><span class="c3 c32">10 mL syringes</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">3 filter sterilization membrane </span>
+                     <p class="c38 c34"><span class="c3 c32">Filter sterilization membrane </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">Distilled/non distilled water</span>
+                     <p class="c38 c34"><span class="c3 c32">Distilled/non distilled water</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">Sodium chloride</span>
+                     <p class="c38 c34"><span class="c3 c32">NaCl</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">Hygromycin</span>
+                     <p class="c38 c34"><span class="c3 c32">Hygromycin (20 mg/mL)</span>
                      </p>
-                     <p class="c6"><span class="c3">20 mL overnight culture (</span><span class="c3 c15">B. subtilis </span><span class="c3">strain</span><span class="c36 c26 c3 c19 c15">) </span>
+                     <p class="c38 c34"><span class="c3 c32">20 mL overnight culture (<i>B. subtilis </i>WB800) </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">LB media </span>
+                     <p class="c38 c34"><span class="c3 c32">LB media </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">3 250 mL erlenmeyer flasks </span>
+                     <p class="c38 c34"><span class="c3 c32">250 mL erlenmeyer flasks</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">Chloramphenicol plates </span>
+                     <p class="c38 c34"><span class="c3 c32">Chloramphenicol agar plates </span>
                      </p>
                  </td>
@@ Line 6,695: / Line 6,695: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c13 c4 c3">Preparation</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Preparation</b></span>
                      </p>
                      <ol class="c10 lst-kix_wfo24hevzk6g-0 start" start="1">
-                         <li class="c6 c11"><span class="c9 c3">Prepare 500 mL of 0.9% W/V saline solution using sodium chloride at a pH of 7.35. Add appropriate antibiotics. Cover using parafilm and store at room temperature</span>
+                         <li class="c8"><span class="c3 c32">Prepare 500 mL of 0.9% W/V saline solution using sodium chloride at a pH of 7.35. Add appropriate antibiotics. Cover using parafilm and store at room temperature.</span>
                          </li>
-                         <li class="c6 c11"><span class="c3">Start 20 mL overnight culture </span><span class="c3 c15">B. subtilis</span>
+                         <li class="c8"><span class="c3 c32">Start 20 mL overnight culture <i>B. subtilis</i>.</span>
                          </li>
-                         <li class="c6 c11"><span class="c3">For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of </span><span class="c3 c15">B. subtilis </span>
+                         <li class="c8"><span class="c3 c32">For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of <i>B. subtilis</i>.
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.</span>
+                         <li class="c8"><span class="c3 c32">Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Repeat process using cell culture in distilled water</span>
+                         <li class="c8"><span class="c3 c32">Repeat process using cell culture in distilled water.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay</span>
+                         <li class="c8"><span class="c3 c32">Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got</span>
+                         <li class="c8"><span class="c3 c32">As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got.</span>
                          </li>
                      </ol>
-                     <p class="c6"><span class="c13 c4 c3">Measurements</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Measurements</b></span>
                      </p>
                      <ol class="c10 lst-kix_i1031kzh8e9u-0 start" start="1">
-                         <li class="c6 c11"><span class="c9 c3">Take up 5 mL of each overnight culture into two 10 mL syringes. Take up 5 mL chloramphenicol LB media into one 10 mL syringe. Attach filter sterilization membrane to one of the syringes containing the 5 mL overnight culture and to the syringe containing 5 mL chlor + LB media </span>
+                         <li class="c8"><span class="c3 c32">Take up 5 mL of each overnight culture into two 10 mL syringes. Take up 5 mL chloramphenicol LB media into one 10 mL syringe. Attach filter sterilization membrane to one of the syringes containing the 5 mL overnight culture and to the syringe containing 5 mL chlor + LB media. </span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">With the 250 mL erlenmeyer flasks, add 10 mL of saline solution</span>
+                         <li class="c8"><span class="c3 c32">With the 250 mL erlenmeyer flasks, add 10 mL of saline solution.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Submerse each of the 10 mL filter sterilization syringes into the 250 mL erlenmeyer flasks. Cover the top of the erlenmeyer flasks with parafilm</span>
+                         <li class="c8"><span class="c3 c32">Submerse each of the 10 mL filter sterilization syringes into the 250 mL erlenmeyer flasks. Cover the top of the erlenmeyer flasks with Parafilm.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">After a 24 hour period take a 1 mL sample of the saline solution in the 250 mL erlenmeyer flasks</span>
+                         <li class="c8"><span class="c3 c32">After a 24-hour period take a 1 mL sample of the saline solution in the 250 mL erlenmeyer flasks.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm </span>
+                         <li class="c8"><span class="c3 c32">Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Take a 150 &micro;L sample of the saline solution and plate it on respective antibiotic plate</span>
+                         <li class="c8"><span class="c3 c32">Take a 150 &micro;L sample of the saline solution and plate it on respective antibiotic plate.</span>
                          </li>
-                         <li class="c6 c11"><span class="c9 c3">Repeat for seven days </span>
+                         <li class="c8"><span class="c3 c32">Repeat once daily for seven days.</span>
                          </li>
                      </ol>
@@ Line 6,755: / Line 6,755: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c9 c3">The purpose of this assay was to determine quantitatively if the backing layer will allow cell growth similar to those found under optimal conditions. </span>
+                     <p class="c38 c34"><span class="c3 c32">The purpose of this assay was to determine quantitatively if the backing layer will allow cell growth similar to those found under optimal conditions. </span>
                      </p>
                  </td>
@@ Line 6,765: / Line 6,765: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c9 c3">1 culture tube</span>
+                     <p class="c38 c34"><span class="c3 c32">10 mL culture tube</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">CoTran 9722 backing layer </span>
+                     <p class="c38 c34"><span class="c3 c32">CoTran 9722 backing layer </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">3 50 mL falcon tubes</span>
+                     <p class="c38 c34"><span class="c3 c32">50 mL falcon tubes</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">LB media with hygromycin</span>
+                     <p class="c38 c34"><span class="c3 c32">LB media with hygromycin</span>
                      </p>
-                     <p class="c6"><span class="c9 c3">Hygromycin agar plates</span>
+                     <p class="c38 c34"><span class="c3 c32">Hygromycin agar plates</span>
                      </p>
                  </td>
@@ Line 6,783: / Line 6,783: @@
                  </td>
                  <td class="c17" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c4 c3 c30">Preparation of </span><span class="c4 c3 c30 c15">B. subtilis </span><span class="c13 c4 c3">WB800 Cells</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Preparation of <i>B. subtilis</i> WB800 Cells</b></span>
                      </p>
                      <ol class="c10 lst-kix_2in1h2fvxudb-0 start" start="1">
-                         <li class="c0"><span class="c3">Two days prior to measuring growth, prepare an overnight culture of </span><span class="c3 c15">B. subtilis </span><span class="c3">in 10 mL LB broth with the appropriate antibiotic (100 </span><span class="c12 c3">&mu;</span><span class="c9 c3">g/mL final concentration of hygromycin). Incubate at 37&deg;C, shaking at 200 rpm overnight.</span>
+                         <li class="c8"><span class="c3 c32">Two days prior to measuring growth, prepare an overnight culture of <i>B. subtilis<i> in 10 mL LB broth with the appropriate antibiotic (100 &mu;g/mL final concentration of hygromycin). Incubate at 37&deg;C, shaking at 200 rpm overnight.</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Inoculation</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Inoculation</b></span>
                      </p>
                      <ol class="c10 lst-kix_7cr24e9okmfb-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35&deg;C, 22&deg;C or 4&deg;C) with lids loosened, shaking at 200 rpm.</span>
+                         <li class="c8"><span class="c3 c32">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35&deg;C, 22&deg;C or 4&deg;C) with lids loosened, shaking at 200 rpm.</span>
                          </li>
                      </ol>
                      <ol class="c10 lst-kix_2in1h2fvxudb-0" start="2">
-                         <li class="c0"><span class="c9 c3">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Measurement of Growth</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Measurement of Growth</b></span>
                      </p>
                      <ol class="c10 lst-kix_v96mlbro49yx-0 start" start="1">
-                         <li class="c0"><span class="c3">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD</span><span class="c7">600</span><span class="c9 c3">). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once!</span>
+                         <li class="c8"><span class="c3 c32">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD<sub>600</sub>). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once!</span>
                          </li>
-                         <li class="c0"><span class="c3">Commence measurement of samples at 8:00 (OD</span><span class="c7">600</span><span class="c3">). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span>
+                         <li class="c8"><span class="c3 c32">Commence measurement of samples at 8:00 (OD<sub>600</sub>). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span>
                          </li>
-                         <li class="c6 c11"><span class="c12 c3">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. </span><span class="c3">&nbsp;If samples reach an OD</span><span class="c7">600</span><span class="c3">&nbsp;&gt; 0.4, dilute the sample 10X using LB-broth with 100 </span><span class="c12 c3">&mu;g/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.</span>
+                         <li class="c8"><span class="c3 c32">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD<sub>600</sub> >0.4, dilute the sample 10X using LB-broth with 100 &mu;g/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.</span>
                          </li>
                      </ol>
@@ Line 6,835: / Line 6,835: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Following the first backing layer growth curve assay, we consulted our mentors and determined that the assay was not effective in determining whether our backing layer would be a limiting factor for gas exchange. This is due to the fact that there is 10x the volume of oxygen in the culture tube to begin with. Therefore, the rate of gas exchange through the backing layer will be negligible. </span>
+                     <p class="c38 c34"><span class="c3 c32">Following the first backing layer growth curve assay, we consulted our mentors and determined that the assay was not effective in determining whether our backing layer would be a limiting factor for gas exchange. This is due to the fact that there is 10x the volume of oxygen in the culture tube to begin with. Therefore, the rate of gas exchange through the backing layer will be negligible. </span>
                      </p>
                      <p class="c2 c28"><span class="c9 c3"></span>
                      </p>
-                     <p class="c2"><span class="c9 c3">This purpose of this assay was to determine whether the bacterial cells can be starved of oxygen with our backing layer using a different protocol. </span>
+                     <p class="c38 c34"><span class="c3 c32">This purpose of this assay was to determine whether the bacterial cells can be starved of oxygen with our backing layer using a different protocol. </span>
                      </p>
                  </td>
@@ Line 6,849: / Line 6,849: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c6"><span class="c9 c3">1 culture tube</span>
+                     <p class="c38 c34"><span class="c3 c32">10 mL culture tube</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">12 2x2 well nuclon plates </span>
+                     <p class="c38 c34"><span class="c3 c32">2x2 well nuclon plates </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">CoTran 9722 backing layer </span>
+                     <p class="c38 c34"><span class="c3 c32">CoTran 9722 backing layer </span>
                      </p>
-                     <p class="c6"><span class="c9 c3">CoTran 9719 backing layer </span>
+                     <p class="c38 c34"><span class="c3 c32">CoTran 9719 backing layer </span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Parafilm </span>
+                     <p class="c38 c34"><span class="c3 c32">Parafilm </span>
                      </p>
                  </td>
@@ Line 6,867: / Line 6,867: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c13 c4 c3">Day Zero</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Day Zero</b></span>
                      </p>
                      <ol class="c10 lst-kix_ghlmud29dl0e-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">Using sterilized 2x2 culture plates, add 1.62 mL of LB media + appropriate antibiotic.</span>
+                         <li class="c8"><span class="c3 c32">Using sterilized 2x2 culture plates, add 1.62 mL of LB media + appropriate antibiotic.</span>
                          </li>
-                         <li class="c0"><span class="c3">Add 180 &micro;L O/N culture that has already been OD</span><span class="c3 c7">600</span><span class="c9 c3">. This will be the baseline reading.</span>
+                         <li class="c8"><span class="c3 c32">Add 180 &micro;L O/N culture that has already been OD<sub>600</sub>. This will be the baseline reading.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Pipette 180 &micro;L of the mixture out, discard.</span>
+                         <li class="c8"><span class="c3 c32">Pipette 180 &micro;L of the mixture out, discard.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Place appropriate backing layer on top of the culture plates. Secure backing layer with parafilm on top criss cross and gain around the edges. </span>
+                         <li class="c8"><span class="c3 c32">Place appropriate backing layer on top of the culture plates. Secure backing layer with Parafilm on top criss cross and gain around the edges. </span>
                          </li>
-                         <li class="c0"><span class="c9 c3">For a positive control, cover culture plate with parafilm. Puncture a hole into each of the wells using a syringe needle. </span>
+                         <li class="c8"><span class="c3 c32">For a positive control, cover culture plate with Parafilm. Puncture a hole into each of the wells using a syringe needle. </span>
                          </li>
-                         <li class="c0"><span class="c9 c3">For a negative control, place the lid on top and parafilm to secure.</span>
+                         <li class="c8"><span class="c3 c32">For a negative control, place the lid on top and Parafilm to secure.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Place in incubator at 35&deg;C at 10 rpm.</span>
+                         <li class="c8"><span class="c3 c32">Place in incubator at 35&deg;C at 10 rpm.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat for replicates. </span>
+                         <li class="c8"><span class="c3 c32">Repeat for replicates. </span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Day One</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Day One</b></span>
                      </p>
                      <ol class="c10 lst-kix_taz15jv9qurq-0 start" start="1">
-                         <li class="c0"><span class="c3">Begin OD</span><span class="c3 c7">600</span><span class="c9 c3">&nbsp;readings using LB + antibiotics. </span>
+                         <li class="c8"><span class="c3 c32">Begin OD<sub>600</sub> readings using LB + antibiotics. </span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Take the reading of the day one plates</span>
+                         <li class="c8"><span class="c3 c32">Take the reading of the day one plates.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Repeat for all membranes and controls </span>
+                         <li class="c8"><span class="c3 c32">Repeat for all membranes and controls </span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Day Three</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Day Three</b></span>
                      </p>
                      <ol class="c10 lst-kix_ma14gllav8iv-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">Repeat day one procedure using day three plates</span>
+                         <li class="c8"><span class="c3 c32">Repeat day one procedure using day three plates</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c13 c4 c3">Day Seven</span>
+                     <p class="c38 c34"><span class="c3 c32"><b>Day Seven</b></span>
                      </p>
                      <ol class="c10 lst-kix_hd8gz1vih14v-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">Repeat day one procedure using day seven plates </span>
+                         <li class="c8"><span class="c3 c32">Repeat day one procedure using day seven plates </span>
                          </li>
                      </ol>
@@ Line 6,933: / Line 6,933: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">With the tireless support of Dr. Craig Jenne, the team wrote and submitted an extensive ethics application to the Health Sciences Animal Care Committee (HSACC) which evaluated the application on multiple grounds such as feasibility, morality, precautions, etc. of the testing of our transdermal patch on mice models. Our protocols and the scientific significance of our transdermal patch were reviewed and approved by the HSACC. </span><span class="c3 c44">&nbsp;</span>
+                     <p class="c38 c34"><span class="c3 c32">With the tireless support of Dr. Craig Jenne, the team wrote and submitted an extensive ethics application to the Health Sciences Animal Care Committee (HSACC) which evaluated the application on multiple grounds such as feasibility, morality, precautions, etc. of the testing of our transdermal patch on mice models. Our protocols and the scientific significance of our transdermal patch were reviewed and approved by the HSACC. </span>
                      </p>
                  </td>
@@ Line 6,943: / Line 6,943: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">This experiment allowed us to examine the effects of the prototype version of our transdermal patch</span><span class="c3 c15">&nbsp;in vivo</span><span class="c9 c3">. This experiment addresses three main aims: </span>
+                     <p class="c38 c34"><span class="c3 c32">This experiment allowed us to examine the effects of the prototype version of our transdermal patch <i>in vivo</i>. This experiment addresses three main aims: </span>
                      </p>
                      <ol class="c10 lst-kix_bg6npkoi2682-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">To determine whether patches containing TD1 tagged mBBI diffused from the patch into systemic circulation of mice. </span>
+                         <li class="c0"><span class="c3 c32">To determine whether patches containing TD1 tagged mBBI diffused from the patch into systemic circulation of mice. </span>
                          </li>
-                         <li class="c0"><span class="c3">To determine whether the membrane of the patch was capable of containing </span><span class="c3 c15">B.subtilis</span><span class="c9 c3">&nbsp;without leakage. </span>
+                         <li class="c0"><span class="c3 c32">To determine whether the membrane of the patch was capable of containing <i>B. subtilis</i> without leakage. </span>
                          </li>
-                         <li class="c0"><span class="c3">To determine whether the adhesive, the peptide or the </span><span class="c3 c15">B.subtilis</span><span class="c9 c3">&nbsp;is immunogenic. </span>
+                         <li class="c0"><span class="c3 c32">To determine whether the adhesive, the peptide or the <i>B. subtilis</i> is immunogenic. </span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c3">Three cohorts of six mice each were used for this experiment. The first cohort was given patches containing water to examine the effects of the adhesive on the skin. In the second cohort, mice were given a patch containing </span><span class="c3 c15">B.subtilis</span><span class="c9 c3">. The third group was used to examine the diffusion of fusion protein across the skin. In order to measure the effects of these patches, the skin samples were obtained post-euthanasia and tested for any immunological responses (ie. neutrophil deposition). The blood samples were collected to check for mBBI presence using mass spectrometry. The blood samples collected were either isolated for serum or for plasma. Since it wasn&rsquo;t known where mBBI would be found, both procedures were conducted to ensure that mBBI can be found in either of those samples. </span>
+                     <p class="c38 c34"><span class="c3 c32">Three cohorts of six mice each were used for this experiment. The first cohort was given patches containing water to examine the effects of the adhesive on the skin. In the second cohort, mice were given a patch containing <i>B. subtilis</i>. The third group was used to examine the diffusion of fusion protein across the skin. In order to measure the effects of these patches, the skin samples were obtained post-euthanasia and tested for any immunological responses (ie. neutrophil deposition). The blood samples were collected to check for mBBI presence using mass spectrometry. The blood samples collected were either isolated for serum or for plasma. Since it was unknown where mBBI would be found, both procedures were conducted. </span>
                      </p>
                  </td>
@@ Line 6,963: / Line 6,963: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2 c47"><span class="c3">18 BALB/c adult mice<br> 18 transdermal patches with respective contents<br> </span><span class="c3">General Anesthesia </span><span class="c3"><br> Hair removal cream<br> </span><span class="c3">Tissue samples stuff<br> Mass spec stuff</span>
+                     <p class="c38 c34"><span class="c3 c32">18 BALB/c adult mice</span></p>
+<p class="c38 c34"><span class="c3 c32">18 transdermal patches with respective contents</span></p>
+<p class="c38 c34"><span class="c3 c32">General Anesthesia</span></p>
+<p class="c38 c34"><span class="c3 c32">Hair removal cream</p></span>
+<p class="c38 c34"><span class="c3 c32">0.5 M EDTA</p></span>
                      </p>
                  </td>
@@ Line 6,976: / Line 6,980: @@
                      </p>
                      <ol class="c10 lst-kix_yuxi9iaet81w-0 start" start="1">
-                         <li class="c0"><span class="c3">Prepare patches with their respective contents in the protocol described her</span><span class="c3">e</span><span class="c9 c3">. Manufacture at least 6 working patches of each type.</span>
+                         <li class="c8"><span class="c3 c32">Prepare patches with their respective contents in the protocol described here. Manufacture at least 6 working patches of each type.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Use 6 mice for treatment - for each patch type</span>
+                         <li class="c8"><span class="c3 c32">Use 6 mice for treatment, one for each patch type.</span>
                          </li>
-                         <li class="c0"><span class="c3">Anesthetize mice with 300 uL of general anesthesia, which made the mice unconscious for roughly 45min. The anesthesia was administered to 6 mice at a tim</span><span class="c9 c3">e.</span>
+                         <li class="c8"><span class="c3 c32">Anesthetize mice with 300 uL of general anesthesia. The anesthesia was administered to 6 mice at a time.</span><span class="c9 c3">e.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Once the mice were under the effects of anesthesia, a hair removal cream (Nair) was used to remove the hair from underneath the shoulder of the mice.</span>
+                         <li class="c8"><span class="c3 c32">Remove hair using hair removal cream (Nair) underneath the shoulder of the mice.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">The patches were applied to the mice on the top of lumbar vertebrae using tissue glue to ensure proper attachment. After the application, the mice were left in the cage for recuperation.</span>
+                         <li class="c8"><span class="c3 c32">Apply one patch to one mouse on the top of lumbar vertebrae using tissue glue to ensure proper attachment. After the application, leave mice caged.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Steps 3-5 were repeated for the mice in the other groups.</span>
+                         <li class="c8"><span class="c3 c32">Repeat Steps 3-5 for other mice in other patch groups.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Once the application was done, they were kept under constant observation to ensure that patch stays on.</span>
+                         <li class="c8"><span class="c3 c32">Keep mice under constant observation to ensure that patch stays on.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">After day 2, three mice from each group were brought into the lab. The mice were anesthetized with 600uL of general anesthesia. (Note: This amount of anesthesia is used when the mice are going to be sacrificed in the immediate future.)</span>
+                         <li class="c8"><span class="c3 c32">After day 2, anesthetize three mice with 600uL of general anesthesia each. (Note: This amount of anesthesia is used when the mice will be sacrificed in the immediate future.)</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Once they are unconscious, blood samples from two of the mice were drawn from the left ventricle to obtain blood serum. Blood serum was obtained by leaving the blood sample in room temp for half hour and when once it coagulates it is isolated by spinning down the sample.</span>
+                         <li class="c8"><span class="c3 c32">Once they are unconscious, draw blood samples two of the mice via the left ventricle to obtain blood. Leave the blood sample at room temperature for half an hour, and upon coagulation, isolate serum by centrifugation.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">The other mouse was then anesthetized, and blood samples were collected using an anticoagulant (EDTA) in order to obtain blood plasma. Once the blood is mixed with the anticoagulant, the plasma is isolated by spinning down the sample.</span>
+                         <li class="c8"><span class="c3 c32">Anesthetize the remaining mouse and collect samples using 8 uL EDTA in order to obtain blood plasma. Once the blood is mixed with the anticoagulant, the plasma is isolated by spinning down the sample.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">After the blood drawing, mouse were sacrificed using cervical dislocation. Skin samples were then obtained from the lumbar region. </span>
+                         <li class="c8"><span class="c3 c32">Sacrifice the mice via cervical dislocation. Obtain skin samples from the lumbar region. </span>
                          </li>
-                         <li class="c0"><span class="c9 c3">At day 5, steps 8-11 were repeated. However, this time two blood samples were collected and blood plasma was isolated. And the third sample was used to obtain blood serum.</span>
+                         <li class="c8"><span class="c3 c32">At day 5, repeat Steps 8-11.</span>
                          </li>
                      </ol>
@@ Line 7,025: / Line 7,029: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c3">Mass spectrometry was used to determine the presence of mBBI in transformed </span><span class="c3 c15">B. subtilis </span><span class="c9 c3">cell lysate and mouse trial blood samples.</span>
+                     <p class="c38 c34"><span class="c3 c32">Mass spectrometry was used to determine the presence of mBBI in mouse trial blood samples.</span>
                      </p>
                  </td>
@@ Line 7,035: / Line 7,039: @@
                  </td>
                  <td class="c31" colspan="1" rowspan="1">
-                     <p class="c2"><span class="c9 c3">Cell lysate or mouse blood sample</span>
+                     <p class="c38 c34"><span class="c3 c32">Mouse blood sample</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Heavy isotope of mBBI peptide</span>
+                     <p class="c38 c34"><span class="c3 c32">Heavy isotope of mBBI peptide</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">C18 Zip-Tip column from Thermo</span>
+                     <p class="c38 c34"><span class="c3 c32">C18 Zip-Tip column from Thermo</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">50% acetonitrile</span>
+                     <p class="c38 c34"><span class="c3 c32">50% acetonitrile</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">0.1% formic acid</span>
+                     <p class="c38 c34"><span class="c3 c32">0.1% formic acid</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">14 cm C18 column</span>
+                     <p class="c38 c34"><span class="c3 c32">14 cm C18 column</span>
                      </p>
-                     <p class="c2"><span class="c9 c3">Mass spectrometer</span>
+                     <p class="c38 c34"><span class="c3 c32">Mass spectrometer</span>
                      </p>
                  </td>
@@ Line 7,058: / Line 7,062: @@
                  <td class="c31" colspan="1" rowspan="1">
                      <ol class="c10 lst-kix_qsfm83ibxmjm-0 start" start="1">
-                         <li class="c0"><span class="c9 c3">Extract peptides from mouse blood plasma or serum via purification on a C18 Zip-Tip column from Thermo; elute with 50% acetonitrile, 0.1% formic acid.</span>
+                         <li class="c8"><span class="c3 c32">Extract peptides from mouse blood plasma or serum via purification on a C18 Zip-Tip column from Thermo; elute with 50% acetonitrile, 0.1% formic acid.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Concentrate samples 10x via evaporation and resuspend in 0.1% formic acid.</span>
+                         <li class="c8"><span class="c3 c32">Concentrate samples 10x via evaporation and resuspend in 0.1% formic acid.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Inject extracted samples onto a 10 cm C18 column and elute with a 30 minute 5-40%B gradient at 300 nL/min.</span>
+                         <li class="c8"><span class="c3 c32">Inject extracted samples onto a 10 cm C18 column and elute with a 30 minute 5-40%B gradient at 300 nL/min.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Acquire MS spectra on a Thermo LTQ Orbitrap Velos in ion trap mode or in Orbitrap mode with CID.</span>
+                         <li class="c8"><span class="c3 c32">Acquire MS spectra on a Thermo LTQ Orbitrap Velos in ion trap mode or in Orbitrap mode with CID.</span>
                          </li>
-                         <li class="c0"><span class="c9 c3">Analyze spectra manually for the presence of mBBI (monoisotopic mass = 2577.1 Da) at various charge states.</span>
+                         <li class="c8"><span class="c3 c32">Analyze spectra manually for the presence of mBBI (monoisotopic mass = 2577.1 Da) at various charge states.</span>
                          </li>
                      </ol>
-                     <p class="c2"><span class="c9 c3">Note: Pure sample of mBBI, synthesized by BioBasic, was used as a reference for identification.</span>
+                     <p class="c38 c34"><span class="c3 c32">Note: Pure sample of mBBI, synthesized by BioBasic, was used as a reference for identification.</span>
                      </p>
                  </td>
@@ Line 7,075: / Line 7,079: @@
          </tbody>
      </table>
      <p class="c14"><span class="c3"></span>
      </p>
      <p class="c14"><span class="c3"></span>
      </p>
+<a id="t.971ab58709574b3dabced61ce6003ae4e49580cb"></a>
+    <a id="t.0"></a>
+    <table class="c23" style="width:625px">
+        <tbody>
+            <tr class="c18">
+                <td class="c25" colspan="2" rowspan="1">
+                    <p class="c2"><span class="c24 c21 c4 c3">Immunohistochemical Staining of Mouse Cryo Cross-Section</span>
+                    </p>
+                </td>
+            </tr>
+            <tr class="c1">
+                <td class="c29" colspan="1" rowspan="1">
+                    <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span>
+                    </p>
+                </td>
+                <td class="c17" colspan="1" rowspan="1">
+                    <p class="c38 c34"><span class="c3 c32">This technique was employed to study any possible immunological responses induced by the transdermal patch, the bacteria <i>Bacillus subtilis</i>, and mBBI. Mice skin tissue samples were obtained from 9 mice at day 2 (samples 1-3 for all treatments) and the rest of the mice samples were obtained on day 5 (samples 4-6 for all treatments) of the study. The skin tissue samples were then fixed, sectioned, and stained. Due to lack of training and materials, members of the Jenne lab had to perform most of the experiment. However, a member of the team periodically assisted or observed them.</span>
+                    </p>
+                </td>
+            </tr>
+            <tr class="c1">
+                <td class="c29" colspan="1" rowspan="1">
+                    <p class="c2"><span class="c16 c4 c3">Materials</span>
+                    </p>
+                </td>
+                <td class="c17" colspan="1" rowspan="1">
+                    <p class="c38 c34"><span class="c3 c32">Formalin </span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Optimal Cutting Temperature (OCT)</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Cryomold</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Blade (VWR Cat #: 95057-832, coated microtone blades)</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Cryostat </span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Staining Glass Container and Acetone </span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">2% BSA+PBS</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Fluorescent mounting medium (Dako, S3023)</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Conjugated Antibody:</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Biolegend FITC anti-mouse Ly6G (clone 1A8), cat#:127606. &nbsp; &nbsp;</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">BD Pharmingen PE anti-mouse CD49b (clone HMalpha), cat#:558759. &nbsp; &nbsp; </span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">eBioscience APC anti-mouse CD45 (clone 30-F11), cat#:17-0451-83. &nbsp;</span>
+                    </p>
+                    <p class="c38 c34"><span class="c3 c32">Fluorescent Microscope </span>
+                    </p>
+                </td>
+            </tr>
+            <tr class="c1">
+                <td class="c29" colspan="1" rowspan="1">
+                    <p class="c2"><span class="c16 c4 c3"><b>Protocol</b></span>
+                    </p>
+                </td>
+                <td class="c6" colspan="1" rowspan="1">
+                    <ol class="c7 lst-kix_ajye8d523edn-0 start" start="1">
+                        <li class="c8"><span class="c3 c32">Tissue samples were collected from the lumbar region of the mice after blood draw and &nbsp; &nbsp;euthanization.</span>
+                        </li>
+                        <p class="c2"><span class="c16 c4 c3"><b>Tissue Fixation </b></span>
+                        </li>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-1 start" start="1">
+                        <p class="c2"><span class="c16 c4 c3">Formalin:</span></p>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-2 start" start="1">
+                        <li class="c8"><span class="c3 c32">Remove the wax that from the application of Nair during the application of the transdermal patch. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Treat tissue with the formalin solution to fix it the skin tissue samples. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Store fixed skin tissue samples -80°C.</span>
+                        </li>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-0" start="3">
+                        <p class="c38 c34"><span class="c3 c32"><b>Cryosection</b> </span>
+                        </p>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-1 start" start="1">
+                        <li class="c8"><span class="c3 c32">Fill an ice box with dry ice, ethanol, and a piece of metal. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Place Optimal Cutting Temperature (OCT) compound and fixed tissue in a cryomold. Ensure the orientation of the tissue is such that all the layers of the tissue are obtained. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Fill cryomold with OCT compound to cover the fixed tissue (approximately 0.5 cm from top of the mold).</span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">When samples are completely frozen,wrap them in individually-labelled foils, seal in plastic bags, and store at -80&deg;C.</span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Put the blade (VWR Cat #: 95057-832, coated microtone blades) in the Cryostat machine and tighten. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Anchor and adjust tissue sample so it is parallel to the blade.</span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Prime the tissue sample and slowly start cutting . </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Allow the section(s) to dry for approximately 3 hrs.</span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Discard the blade. </span>
+                        </li>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-0" start="4">
+                        <p class="c38 c34"><span class="c3 c32"><b>Cryosection FIxation</b></span>
+                        </p>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-1 start" start="1">
+                        <li class="c8"><span class="c3 c32">Insert tissue slide in staining glass container that containing Acetone.</span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Incubate at room temperature for 10 minutes. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Fill staining glass container with the slides with prepared 2% BSA+PBS. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Incubate at room temperature for 2 hours. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Wipe the slide in such a way that a dome shape is created, and added 2% BSA+PBS to the dome. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Add 1 uL of conjugated antibody to the dome. Incubate at room temperature for 1 hr. Ensure to add water to keep the slide moist at all times. Antibodies used are listed below:</span>
+                        </li>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-2 start" start="1">
+                        <li class="c0"><span class="c1">Biolegend FITC anti-mouse Ly6G (clone 1A8), &nbsp;cat#:127606 </span>
+                        </li>
+                        <li class="c0"><span class="c1">BD Pharmingen PE anti-mouse CD49b (clone HMalpha), &nbsp;cat#:558759</span>
+                        </li>
+                        <li class="c0"><span class="c1">eBioscience APC anti-mouse CD45 (clone 30-F11), cat#:17-0451-83 </span>
+                        </li>
+                    </ol>
+                    <ol class="c7 lst-kix_ajye8d523edn-1" start="7">
+                        <li class="c8"><span class="c3 c32">Wash the slide three times with 2% BSA+PBS.</span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Add 1 drop of Fluorescent mounting medium (Dako, S3023) to slide. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Gently load the cover slip. </span>
+                        </li>
+                        <li class="c8"><span class="c3 c32">Keep the tissue slide at 4°C.</span>
+                        </li>
+<li class="c8"><span class="c3 c32">Visualize sample via fluorescent microscopy.</span>
+                        </li>
+                    </ol>
+                </td>
+            </tr>
+        </tbody>
+    </table>
+    <p class="c22 c13"><span class="c10"></span>
+    </p>
+    <p class="c13 c22"><span></span>
+    </p>
      <p class="c14"><span class="c3"></span>
      </p>