Team:MIT/Contribution

Contributions

Overview

  1. Assembling a parts library for B. subtilis

  2. Working with B. subtilis

  3. Debugging potentially toxic inserts

Assembling a parts library for B. subtilis

  1. Our team has built a library of parts and verified a large number of parts to construct a B. subtilis probiotic for treating MSUD. In addition, we constructed several MoClo compatible backbones for integrating parts into B. subtilis. We believe these parts will be highly useful for assaying and testing purposes. The MoClo backbones will be relevant for other applications outside the scope of our project, as they can be used to construct any parts for integration into the B. subtilis genome.
    See our partspage for more details.

Working with B. subtilis

  1. Our mentor from the Grossman lab works extensively with B. subtilis . Therefore, we were able to obtain and adapt many protocols for working with B. subtilis . We believe these protocols will be useful for any future projects related to B. subtilis. Read more on our experimentspage.

Debugging potentially toxic inserts

  1. In our project, two constitutively expressed inserts proved problematic during the cloning process. We obtained very few transformed E. coli colonies containing inserts, all of which would be faulty. We hypothesized that cloning recombinant periplasmic membrane proteins would reduce fitness of the cell. More information is on our engineering success page. In short, we discovered/hypothesized a couple methods to mitigate this.
  2. Plate, incubate, or inoculate cells at 30C to slow growth. This allows the cells to better manage the insert. If that does not suffice, reducing temp to 25-27 (room temperature) should suffice in most cases. Colonies obtained this way will tend to be smaller and take longer to grow (~24-48 hours).
  3. Down-regulate the construct. This can be done by assembling the construct with an inactive promoter that can be flipped on with a recombinase, or a promoter that is non-characteristic in the chassis.
  4. Attempt to clone using commercially available stable cells. Although most strains of commercially available competent E. coli cells have some form of endonuclease, restriction, and/or recombination deficient phenotype, difficulty cloning unstable inserts and plasmids containing repeat elements may still persist. Stable cells typically contain all of the above, in addition to more recombination deficient phenotypes. However, efficiency may remain low, and these cells may still not be viable for cloning recombinant proteins.
  5. One possibility is that our plasmids contain ori sites designating high copy plasmids. We hypothesized that cloning our constructs into lower copy plasmids may suffice to fix toxic inserts; however, we were unable to obtain other plasmids to fix this. Given enough time, we would try this method to see if fitness can be improved.