Difference between revisions of "Team:UofC Calgary/Judging"
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<p class="c-font-white" style="font-size: 12px"><i>B. subtilis</i> is a gram-positive bacterial species widely used in molecular biology labs around the world. It is capable of natural transformation under conditions of nutrient deprivation (energy starvation), and unlike many gram-negative species <i>B. subtilis</i> does not appear to require a specific uptake sequence. However, transformation through starvation may not be the most ideal process to implement in the lab as the protocols are very time-consuming, very sensitive to precise timings, and can be unreliable. Fortunately, all of the DNA uptake genes are under the control of a single transcription factor, so we can bypass the need for energy starvation by using cells derived from a specific strain of <i>B. subtilis</i> (SCK6) with the pAX01-comK plasmid constructed by Zhang (2010). </p> | <p class="c-font-white" style="font-size: 12px"><i>B. subtilis</i> is a gram-positive bacterial species widely used in molecular biology labs around the world. It is capable of natural transformation under conditions of nutrient deprivation (energy starvation), and unlike many gram-negative species <i>B. subtilis</i> does not appear to require a specific uptake sequence. However, transformation through starvation may not be the most ideal process to implement in the lab as the protocols are very time-consuming, very sensitive to precise timings, and can be unreliable. Fortunately, all of the DNA uptake genes are under the control of a single transcription factor, so we can bypass the need for energy starvation by using cells derived from a specific strain of <i>B. subtilis</i> (SCK6) with the pAX01-comK plasmid constructed by Zhang (2010). </p> | ||
<p class="c-font-white" style="font-size: 12px">This part contains a xylose-inducible promoter (pxylA), a ribosome binding site, and the comK coding sequence. This part is meant to be inserted into the genome of <i>B. subtilis</i> using an appropriate integration vector (unfortunately likely requiring the traditional transformation procedure). Once complete, you have a strain that can be transformed quickly and easily.</p> | <p class="c-font-white" style="font-size: 12px">This part contains a xylose-inducible promoter (pxylA), a ribosome binding site, and the comK coding sequence. This part is meant to be inserted into the genome of <i>B. subtilis</i> using an appropriate integration vector (unfortunately likely requiring the traditional transformation procedure). Once complete, you have a strain that can be transformed quickly and easily.</p> | ||
| + | <p class="c-font-white" style="font-size: 12px"> Previous team(s) that have worked with this part: <a href="https://2014.igem.org/Team:Calgary/Project/BsDetector/BsChassis">University of Calgary iGEM 2014</a> | ||
| + | <p class="c-font-white" style="font-size: 12px"> We have modified this part by removing previously existing internal restriction sites as well as adding flanking homology regions to the <i>B. subtilis</i> genome for better transformation efficiency. </p> | ||
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 01:06, 17 October 2016
Bronze Requirements
- Register and Attend
- DELIVERABLES
- Wiki (Home Page)
- Poster
- Presentation
- Sample Submission
- Safety Form
- Judging Forms
- Parts Page
- Attributions
- New Part BBa_K2008003
We have registered for and will be attending the 2016 iGEM Jamboree in Boston!
You can find the contributions of each of our members as well as our numerous amazing mentors on our Attributions page
We designed and characterized a gene construct coding for the radioprotective peptide mBBI attached to a secretion tag, TD1 tag for secretion and transdermal delivery, as well as GFP for easy transformation recognition. Find it on our parts page here
Silver Requirements
- Part Characterization BBa_K2008005
- Collaboration
- Human Practices
This part contains an active nonamer from the Bowman Birk Protease Inhibitor (BBI) with the addition of lysine, serine, cysteine, and isoleucine (KSCI) tag on the N-terminal end and a phenylalanine (F) tag on the C-terminal end to increase BBI solubility. A B. subtilis secretory signal peptide sequence is included to allow for secretion of the peptide directly into surrounding media. A transdermal tag (TD1, BBa_K1074000) is fused to the N-terminus of BBI to allow for diffusion of the peptide across the skin. This coding sequence is under the control of the constitutive B. subtilis promoter pVeg (BBa_K143012) and a strong B. subtilis RBS (BBa_K780001).
We characterized this part by testing the effect of the BBI peptide in cell culturing experiments using both wildtype fibroblasts (1BR3) as well as colon cancer cells (HCT116). We further characterized the delivery capabilities of our TD1 tag by administering patch prototypes to mice, and then performing mass spectrometry on the mice blood a few days after patch administration to detect the presence of our peptide in the mice blood.
We have collaborated with various iGEM teams as well as certain local organizations for the improvement of our project and for the promotion of iGEM. See all of our collaborations on our Collaborations page.
Our project was continually informed by discussions with professionals in the field. See our human practices efforts on our Human Practices pages.
Gold Requirements
- Integrated Human Practices
- Improving upon a previous part BBa_K1444018
See how the feedback we got from professionals fed back into our project design on Human Practices Gold page.
B. subtilis is a gram-positive bacterial species widely used in molecular biology labs around the world. It is capable of natural transformation under conditions of nutrient deprivation (energy starvation), and unlike many gram-negative species B. subtilis does not appear to require a specific uptake sequence. However, transformation through starvation may not be the most ideal process to implement in the lab as the protocols are very time-consuming, very sensitive to precise timings, and can be unreliable. Fortunately, all of the DNA uptake genes are under the control of a single transcription factor, so we can bypass the need for energy starvation by using cells derived from a specific strain of B. subtilis (SCK6) with the pAX01-comK plasmid constructed by Zhang (2010).
This part contains a xylose-inducible promoter (pxylA), a ribosome binding site, and the comK coding sequence. This part is meant to be inserted into the genome of B. subtilis using an appropriate integration vector (unfortunately likely requiring the traditional transformation procedure). Once complete, you have a strain that can be transformed quickly and easily.
Previous team(s) that have worked with this part: University of Calgary iGEM 2014
We have modified this part by removing previously existing internal restriction sites as well as adding flanking homology regions to the B. subtilis genome for better transformation efficiency.
