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

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 BBI, we wanted to study the repair kinetics in cells treated with BBI 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

1. Prep and Irradiation
2. 1.  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..
3. 2.  Treat the confluent plates with BBI (as outlined in Table 1) and incubate at 37℃ with 5% CO2 for 6 hours.
4. 3.   Irradiate plates which are intended for irradiation (outlined in Table 1) with 2 Gy. We used a Gammacell 1000 irradiator (MDS Nordion).
5. Immunofluorescence Staining
6. 4.  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.
7. 5.  Cells on glass coverslips are fixed for 10 minutes by adding 100μl of a 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.
8. 6.  Wash cells twice in 1X PBS and store in 1X PBS until all plates have been fixed at their corresponding time points.
9. 7.  Add 100 μl of 1X PBS solution with 0.2% Triton X100 (v/v) to the cells for 3 minutes to allow permeabilization.
10. 8.  Incubate cells in 100 μl 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.
11. 9.  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.
12. 10. Wash cells 3 times in 1X PBS.
13. 11. Pour 2 mls of 1X PBS containing 0.1 μg/ml DAPI into the  dishes and leave on for 10 mins.
14. 12. Wash cells 3 times in 1X PBS.
15. 13. Microscopy slides were prepared by adding one drop of Vectashield onto the slide.
16. 14. 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.
17. 15. Seal the edges of the coverslip onto the slide by brushing on clear nail polish over the edges.
18. Counting Foci
19. 16. 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.
20. 17. Foci can be counted on an epifluorescent microscope under red and green channels.

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, triggering apoptosis. If the DNA damage can be repaired, the cells survive. To test whether BBI 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 BBI.

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

21. 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.
22. Trypsinize the stock flask of cells to be tested for radiosensitivity.
23. Obtain a cell count, then add 250,000 cells to the 5ml medium in the T-75 flask.
24. Place T-75 flasks with the cell cultures in a 37℃ incubator with 5% CO2, and keep cap to the flask loose to allow gas exchange.
25. Allow the cells to settle and attach as a monolayer.

Irradiation of Flasks and Performance of Plating Experiment for Clonogenic Assay

26. Label all 60 mm plates according to cell line, drug and radiation exposure.
27. Place 5mL of complete medium in each plate
28. Label all 15 ml tubes according to 1:1, 1:10, 1:100 for each Gy dose level (0 and 5).
29. Put 4.5 ml of complete medium into the 1:10 and 1:100 tubes, but not the 1:1 tubes.
30. Ensure that the Coulter counter is set to the appropriate size parameter for the cell line.
31. Irradiate the flasks at the appropriate dose.
32. Starting with the 0 Gy flask, aspirate the medium and then rinse the cells with PBS, then trypsinize.
33. 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.
34. 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

35. Resuspend the cell pellet of the 15 ml tube.
36. Place 0.5 ml of the 1:1 dilution into the 1:10 dilution tubes of both the 0 Gy and 5 Gy tubes.
37. Calculate the volume of the cell solution needed to plate ~50-100 cells.
38. 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.
39. Place all plates into the incubator. Incubate for 14 days at 37C with 5% CO2.

Staining of Plates

40. Take out all plates from the incubator and aspirate media with vacuum
41. Add 1mL fixing solution to each of the plates (3% acetic acid, 8% methanol, 89% dH2O)
42. Allow cells to sit in fix solution for 2 minutes, then aspirate fix solution
43. Add 1mL stain solution to each of the plates (0.2% (w/v) Gentian violet, 10% formalin in PBS)
44. Let plates sit in stain solution for 5 minutes and aspirate stain solution
45. 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.
46. Let the plates air-dry overnight. Count the next day.

Counting of Colonies

47. 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.
48. 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 %).
49. 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%)

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

1. Add 10 uL ddH2O to desired well (it will become red).
2. Pipette up and down.
3. Incubate at room temperature for 10 minutes.
4. 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

1. Centrifuge tube containing DNA for 3-5 seconds at 3000 g, ensuring all material is at the bottom of the tube.
2. Add ddH2O to reach a final concentration of 50 ng/μL.
3. Vortex.
4. Incubate at 50°C for 20 minutes.
5. 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

1. Grow 2.5 mL of transformed culture overnight in Luria-Bertani broth with appropriate antibiotic.
2. Transfer 2 mL to a 2 mL microcentrifuge tube and pellet the cells by spinning at 3500 g for 1 minute. Discard supernatant.
3. Resuspend pellet in 300 μL Resuspension buffer.
4. Add 300 μL Lysis buffer, invert gently and incubate at room temperature for 3-5 minutes.
5. Add 300 μL Precipitation buffer, invert gently. A white precipitate should form.
6. Centrifuge at 14,000 g for 10 minutes at room temperature.
7. Retain supernatant in a clean 1.5 mL microcentrifuge tube.
8. Add 650 μL isopropanol, gently invert and incubate at room temperature for 10 minutes.
9. Centrifuge at 14,000 g for 10 minutes at 4°C. Discard supernatant.
10. Wash pellet with 500 μL cold 70% ethanol. Add to microcentrifuge tube, do not resuspend.
11. Centrifuge at 14,000 g for 5 minutes at 4°C. Discard supernatant.
12. Dry pellet in speed vac for 10 minutes.
13. 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

1. Into a 0.2 mL PCR tube, add the following:

• 1 μg DNA
• 1 uL 100X BSA
• 1 uL restriction enzyme 1
• 1 uL restriction enzyme 2
• 2 uL 10X appropriate buffer
• ddH2O to a total volume of 20 μL

2. Incubate tube at 37°C for 1 hour.
3. 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

1. To vector tube from restriction digest, add:

• 1 μL 10X Antarctic phosphatase buffer
• 1 μL Antarctic phosphatase
• 8 μL ddH2O

2. Incubate tube at 37°C for 30 minutes.
3. 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

1. 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

2. Incubate tube at room temperature overnight.
3. 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

1. Add 4 μL of PCR-grade ddH2O to 0.2 mL PCR tube.
2. 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.
3. To each PCR tube, add:
4. 0.2 μL Taq DNA polymerase
5. 5 μL 10X Buffer A
6. 1 μL 10 mM dNTP
7. 2 μL 10 μM forward primer
8. 2 μL 10 μM reverse primer
9. 35.8 μL PCR-grade ddHcO

Running PCR

10. 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

1. 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.
2. Cover flask loosely with aluminum foil, secure with autoclave tape, and sterilize by autoclaving for 20 minutes.
3. 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.
4. 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

1. 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.
2. 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).
3. Spin down cells in 50 mL Falcon tube at 8200 g for 10 minutes at 4 °C.
4. Resuspend cells in 12.5 mL cold 50 mM CaCl2 and place on ice for 10 minutes.
5. Repeat Step 3.
6. Resuspend cells in 2 mL cold 50 mM CaCl2, 15% glycerol and place on ice for 30 minutes.
7. 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

1. Using aseptic technique, pipette 0.5 mL of 50% sterile glycerol into a 1.5 mL cryo-tube.
2. Using aseptic technique, add 0.5 mL of overnight culture.
3. Pipette up and down gently to mix.
4. 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

1. Using aseptic technique, pipette 50-100 μL of bacterial culture onto antibiotic agar plate.
2. Dip spreading rod in 70% ethanols, pass over flame and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture.
3. Use rod to spread bacterial culture over entire plate, spinning the plate at the same time.
4. Dip spreading rod in 70% ethanol, pass over flame and allow for excess liquid to burn off.
5. 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

1. Using aseptic technique, flame inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.
2. 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.
3. Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.
4. Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1.4 of the surface of the agar.
5. Repeat Steps 3 and 4 two more times.
6. Flame the inoculation loop until red hot. Allow it to cool for 10 seconds.
7. 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

1. 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).
2. Allow flask to cool in fumehood until warm to the touch before adding 5 μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.
3. Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.
4. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.
5. Load samples of 20 μL DNA containing 2 μL loading dye.
6. 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

1. 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

2. 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.
3. Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.
4. 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

5. 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!
6. 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.
7. 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 BBI. 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

1. 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

2. 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.
3. Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.
4. 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

5. 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!
6. 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.
7. 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.
8. 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 B. 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

1. Thaw 200 μL aliquot of competent E. coli TOP10 cells on ice just before use.
2. Add 0.3-1 μg DNA to cells (in maximum 20 μL), flick gently to mix, and place on ice for 30 minutes.
3. Heat shock for 60-75 seconds at 42°C.
4. Place on ice for 5 minutes.
5. Add 250 μL Luria-Bertani medium to aliquot of cells.
6. Incubate cells for 60 minutes at 37°C, shaking at 200 rpm for 1 hour.
7. Pellet cells in a microcentrifuge at 3500 g for 1 min and discard supernatant.
8. Resuspend pellet in 250 μL Luria-Bertani broth.
9. Plate 50-100 μL of resuspended culture on agar plate with appropriate antibiotic and spread.
10. 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 BBI construct for continuous BBI 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

1. For all stock solutions, autoclave solutions for 20 minutes and store at room temperature.
2. 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.
3. 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 BBI construct for continuous BBI 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

4. Streak cells on Luria-Bertani agar plate in the evening at 17:00. Incubate cells at 30°C overnight.
5. 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.
6. Transfer 0.5 mL SP1 culture to 4.5 mL SP2 medium prewarmed to 37°C in a 16x125 mm culture tube. Incubate at 37°C for 1 hour and 30 minutes, shaking at 150 rpm.
7. Add 50 μL 100X EGTA to the SP2 culture and incubate at 37°C for another 10 minutes, shaking at 150 rpm.
8. 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.
9. Plate 0.25 mL of sample on Luria-Bertani agar plate with appropriate antibiotic.
10. Incubate plate at 37°C overnight or until desired growth is observed.

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

3 50 mL falcon tube

3 15 mL falcon tube

Distilled/non distilled water

Sodium chloride

20 mL overnight culture (B. subtilis strain)

LB media

CoTran 9716 and 9728 semipermeable membrane

Parafilm

Protocol

Preparation

1. 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
2. Start 20 mL overnight culture B. sub
3. 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. sub
4. Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.
5. Repeat process using cell culture in distilled water
6. Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay
7. 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

1. Aliquot 5 mL of each overnight culture into three 15 mL falcon tubes
2. With one of the 15 mL falcon tube wrap parafilm over the top of the tube
3. 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
4. With the 50 mL falcon tubes, add 30 mL of saline solution
5. 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
6. After a 24 hour period take a 1 mL sample of the water in the 50 mL falcon tube
7. Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm
8. Repeat 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. Sub WB800 and chloramphenicol for E.coli GFP + BBI. This ensured that there would be no contamination in our samples.  

Materials

3  10 mL syringes

3 filter sterilization membrane

Distilled/non distilled water

Sodium chloride

Hygromycin

20 mL overnight culture (B. subtilis strain)

LB media

3 250 mL erlenmeyer flasks

Chloramphenicol plates

Protocol

Preparation

1. 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
2. Start 20 mL overnight culture B. sub
3. 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. sub
4. Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.
5. Repeat process using cell culture in distilled water
6. Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay
7. 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

1. 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
2. With the 250 mL erlenmeyer flasks, add 10 mL of saline solution
3. Submerse each of the 10 mL filter sterilization syringes into the 250 mL erlenmeyer flasks. Cover the top of the erlenmeyer flasks with parafilm
4. After a 24 hour period take a 1 mL sample of the saline solution in the 250 mL erlenmeyer flasks
5. Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm
6. Take a 150 µL sample of the saline solution and plate it on respective antibiotic plate
7. Repeat 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

1 culture tube

CoTran 9722 backing layer

3 50 mL falcon tubes

LB media with hygromycin

Hygromycin agar plates

Protocol

Preparation of B. subtilis WB800 Cells

1. 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

1. 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.

2. Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.
3. 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

1. 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!
2. 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.
3. 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

1 culture tube

12 2x2 well nuclon plates

CoTran 9722 backing layer

CoTran 9719 backing layer

Parafilm

Protocol

Day Zero

1. Using sterilized 2x2 culture plates, add 1.62 mL of LB media + appropriate antibiotic.
2. Add 180 µL O/N culture that has already been OD600. This will be the baseline reading.
3. Pipette 180 µL of the mixture out, discard.
4. Place appropriate backing layer on top of the culture plates. Secure backing layer with parafilm on top criss cross and gain around the edges.
5. For a positive control, cover culture plate with parafilm. Puncture a hole into each of the wells using a syringe needle.
6. For a negative control, place the lid on top and parafilm to secure.
7. Place in incubator at 35°C at 10 rpm.
8. Repeat for replicates.

Day One

1. Begin OD600 readings using LB + antibiotics.
2. Take the reading of the day one plates
3. Repeat for all membranes and controls

Day Three

1. Repeat day one procedure using day three plates

Day Seven

1. Repeat day one procedure using day seven plates

in vivo Mouse Testing

Ethics

With the tireless support of Dr. Craig Jenne, we submitted an extensive ethics application to the Conjoint Health Research Ethics Board (CHREB), which evaluated the applications on multiple grounds such as feasibility, morality, precautions, etc. of the testing of our transdermal patch on mice models. As well, Health Science Animal Care Committee (HSACC) scientifically reviewed our protocols, the scientific significance of our transdermal patch, and feasibility. HSACC approved our project under the above mentioned parameters.  Furthermore, the protocols and the procedure were evaluated and were deemed moral and approved by Health Sciences Animal Care Committee (ACC).  

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:

1. To determine whether patches containing TD1 tagged BBI diffused from the patch into systemic circulation of mice.
2. To determine whether the membrane of the patch was capable of containing B.subtilis without leakage.
3. 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 neutrophil deposition. The blood samples were collected to check for BBI presence using mass spectrometry. The blood samples collected were either isolated for serum or for plasma. Since it wasn’t known where BBI would be found, both procedures were conducted to ensure that BBI can be found in either of those samples.

Materials

18 BALB/c adult mice
18 transdermal patches with respective contents
General Anesthesia
Hair removal cream
Tissue samples stuff
Mass spec stuff

Protocol

1. Prepare patches with their respective contents in the protocol described here. Manufacture at least 6 working patches of each type.
2. Use 6 mice for treatment - for each patch type
3. 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 time.
4. 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.
5. 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.
6. Steps 3-5 were repeated for the mice in the other groups.
7. Once the application was done, they were kept under constant observation to ensure that patch stays on.
8. 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.)
9. 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.
10. 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.
11. After the blood drawing, mouse were sacrificed using cervical dislocation. Skin samples were then obtained from the lumbar region.
12. 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.

Detection of BBI by Mass Spectrometry

Experimental Details and Rationale

Mass spectrometry was used to determine the presence of BBI in transformed B. subtilis cell lysate and mouse trial blood samples.

Materials

Cell lysate or mouse blood sample

Heavy isotope of BBI peptide

C18 Zip-Tip column from Thermo

50% acetonitrile

0.1% formic acid

14 cm C18 column

Mass spectrometer

Protocol

1. 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.
2. Concentrate samples 10x via evaporation and resuspend in 0.1% formic acid.
3. Inject extracted samples onto a 10 cm C18 column and elute with a 30 minute 5-40%B gradient at 300 nL/min.
4. Acquire MS spectra on a Thermo LTQ Orbitrap Velos in ion trap mode or in Orbitrap mode with CID.
5. Analyze spectra manually for the presence of BBI (monoisotopic mass = 2577.1 Da) at various charge states.

Note: Pure sample of BBI, synthesized by BioBasic, was used as a reference for identification.