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− | <p | + | <p><font size="2">Haling, S. (1997). Super-rich medium. <i>Biochemistry</i>(16), 2280-2884.<br><br> |
− | + | 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. <i>Cell Cycle</i>, 9(4), 662–669.<br><br> | |
− | + | Spizizen, J. (1958). Transformation of biochemically deficient strains of <i>Bacillus subtilis</i> by deoxyribonucleate. <i>Proceedings of the National Academy of Sciences of the United States of America</i>(44), 1072-1078.</size> | |
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Revision as of 15:54, 16 October 2016
![](https://static.igem.org/mediawiki/2016/d/d2/T--UofC_Calgary--project.jpg)
Experiments
BioTarget
Double-Stranded Break Assay using Immunofluorescence |
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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.
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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)
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Protocol |
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Table 1: Experimental Outline of BBI treatment and Ionizing Radiation. BBI treatment was administered at 30uM and cells were irradiated at 2Gy.
Clonogenic Cell Survival Assay |
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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
Irradiation of Flasks and Performance of Plating Experiment for Clonogenic Assay
Serial Dilutions and Incubation
Staining of Plates
Counting of Colonies
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CHASSIS
Rehydration of Registry DNA |
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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 |
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Rehydration of IDT Synthesized DNA |
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Experimental Details and Rationale |
We ordered genetic constructs from IDT to clone into the pSB1c3 backbone. |
Materials |
Synthesized DNA from IDT ddH2O |
Protocol |
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Plasmid MiniPrep from Escherichia coli and Bacillus subtilis |
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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)
Lysis buffer
Precipitation buffer
Isopropanol 70% ethanol Table-top centrifuge 2 mL microcentrifuge tubes 1.5 mL microcentrifuge tubes |
Protocol |
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Restriction Digest |
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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 |
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Antarctic Phosphatase Treatment |
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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 |
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Ligation of DNA Inserts to Plasmid Backbones |
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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 |
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Colony PCR for Escherichia coli and Bacillus subtilis using the KAPA HiFi PCR Kit |
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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
Running PCR
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Preparation of Agar with Antibiotics |
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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
Appropriate antibiotic
1500 mL Erlenmeyer flask |
Protocol |
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Preparation of Chemically Competent Escherichia coli Cells |
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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
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 |
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Glycerol Stock Preparation of Transformed Escherichia coli and Bacillus subtilis |
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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 |
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Plating Culture Broth on Agar Plates |
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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 |
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Streaking of Agar Plates |
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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 |
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Agarose Gel Electrophoresis |
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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
Agarose 250 mL Erlenmeyer flask RedSafe Nucleic Acid Staining Solution Gel casting tray and comb 10X loading dye DNA sample Microwave |
Protocol |
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Growth Curve of Bacillus subtilis: Temperature Simulation |
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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
Bacillus subtilis WB800 cells 16x125 mm culture tubes 50 mL Falcon tubes Spectrophotometer 1 mL cuvette |
Protocol |
Preparation of B. subtilis WB800 Cells
Inoculation
Measurement of Growth
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Growth Curve of Bacillus subtilis: Media Optimization |
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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
2X Luria-Bertani broth medium with hygromycin
Super Rich broth medium
Bacillus subtilis WB800 cells 16x125 mm culture tubes 50 mL Falcon tubes Spectrophotometer 1 mL cuvette |
Protocol |
Preparation of B. subtilis WB800 Cells
Inoculation
Measurement of Growth
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Transformation of Escherichia coli TOP10 |
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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
Agar plate with appropriate antibiotic |
Protocol |
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Preparation of SP1 and SP2 Media for Bacillus subtilis WB800 Transformation (adapted from Spizizen, 1958) |
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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
50 mM CaCl2 250 mM MgCl2 50% filter-sterilized glucose Casamino acids/yeast extract
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Protocol |
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Preparation of Bacillus subtilis WB800 Competent Cells and Transformation |
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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 |
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DEVICE
Semipermeable Membrane Diffusion Assay |
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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
Measurements
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Filter Sterilization Membrane Diffusion Assay |
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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
Measurements
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Backing Layer Growth Curve Assay |
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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
Inoculation
Measurement of Growth
|
Backing Layer Survival Assay |
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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
Day One
Day Three
Day Seven
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in vivo Mouse Testing |
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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:
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 |
Protocol |
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Detection of BBI by Mass Spectrometry |
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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 |
Note: Pure sample of BBI, synthesized by BioBasic, was used as a reference for identification. |
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.