Difference between revisions of "Part:BBa K1692028"

Latest revision as of 20:41, 18 September 2015

cotZ-aeBlue-CIPA

The goal of our project BioHYDRA was to replace all the parts of HYDRAs by biologically produced substances. We sought out to replace polyamide tape by bacterially cellulose, and the glue by cellulose binding domains on the surface of the spore coat. Thus, the first step involved cloning a Bacillus subtilis construct in Escherichia coli of a fusion protein sequencing consisting of a spore coat protein, cotZ (building off work done on Sporobeads by the LMU Munich 2012 iGEM team), and a cellulose binding domain (CIPA). Additionally, we decided to add aeBlue, a chromogenic protein, between cotZ and CIPA to be able to see with the naked eye whether B. subtilis is in a vegetative or a spore state.

BioHYDRA: We were able to successfully create the fusion protein cotZ-aeBLUE-CIPA in E. coli . This construct was ligated into the pSBbs1C backbone (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K823023">BBa_K823023</a>). We then transformed E. coli to grow our plasmid. We are excited to have been able to use our newly developed <a href="http://2015.igem.org/Team:Stanford-Brown/CRATER"> CRATER </a> technique to better select for our plasmid when transforming, which accelerated our project substantially. By running the plasmid through a gel and by sequencing, we were able to confirm that we had the right size (8 kb) and sequence for the plasmid.
We then transformed our construct into B. Subtilis. This was our main setback for this project, as many protocols did not seem to function. We believe that our 8 kb construct was very large and thus was hard to transform. After trying Xylose competence induced cells and electroporation, we decided to use the LMU Munich's MNGE transformation protocol. by picking certain colonies and undergoing colony PCR, we found that certain colonies seem to contain our part, which was confirmed by sequencing. Below is some data showing differential absorption between our control (aeBlue-) and our two samples which contain the insert (aeBlue+), using a spectrophotometer.
<img src="" alt="Generic placeholder image">
Pic 5. Absorption spectra of our transformed spores (aeBlue+) and wild type spores (aeBlue-).

While absorbance at 597 nm (characteristic to <a href="https://parts.igem.org/Part:BBa_K1033929"> aeBlue</a>) does not show, there is a higher overall absorbance despite having the same spore concentration. Further testing is being done to determine the cause of this absorbance.

We also undertook a cellulose binding assay using scanning electron microscopy. We used four samples:
1. Wild type spores without a PBS wash
2. Spores with our contstruct without a PBS wash
3. Wild type spores with a PBS wash
4. Spores with our contstruct with a PBS wash
Here are our results:
<img src="" alt="Generic placeholder image">
Pic. 6 a. Wild type spores without a PBS wash (35 cells). b. Wild type spores with a PBS wash (10 cells). c. Spores with our construct without a PBS wash (463 cells). d. Spores with our construct with a PBS wash(93 cells).

We can see that there is not only a net difference between washing and not washing the cellulose. But even more importantly, we can see that our construct yields a greater amount of spores adhering to the cellulose. These pictures were taken as representative of each sample, and thus these preliminary cell counts are accurate enough to show a statistical difference between cells that contain our construct and cells that do not. This shows that our construct works.

Sequence and Features