Team:MichiganState/Design/HGT

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Project Design

General Anoxic HGT Biosensor Bioreactor Design

When designing the toxin-antitoxin system, we first started with finding two plasmids with different origins of replication, so that they would both work inside the same cell, pCM80, and pAWP78. The toxin, ghoT, is relatively small and might be hard to see on a gel, so we designed the insert with overhangs in the middle of a LacZ gene on pCM80. This would make it possible to see if the insert was there using a blue-white screening. Also, when designing the toxin insert to be synthesized, we added a cumate inducible promoter, CuO, so that the toxin would not be transcribed and kill the cell unless induced by cumate. For the antitoxin, we used a medium promoter in E. coli, pmoa, and had it synthesized with overhangs on the pAWP78 plasmid, so that we could perform a Gibson assembly.

Figure 1a (left): The ghoT construct that is the toxin inserted into the LacZ gene of the pCM80 plasmid. Figure 1b (right): The ghoS construct with the antitoxin inserted into the pAWP78 plasmid. Both images were taken from benchling.com

In order to test the for the ghoT + pCM80 construct we transformed them into E. coli and plated them on X-gal/IPTG/Tetracycline LB plates that allow us to do a blue-white screening of the colonies. Once we pick the white colonies, the colonies that were properly transformed, of the plates we will grow them in LB/Tetracycline broth in a plate reader with varying amounts of cumate. The cumate will theoretically activate the promoter and the toxin will be produced, which we will see based on the OD of the cultures after cumate is added.

The ghoS + pAWP78 construct will be checked for the insert via colony PCR. Once the construct is complete it will be transformed into E. cloni 10G with the ghoT toxin plasmid and then grown on a plate reader to see if the effects of the toxin are negated.

After both constructs are working, the E. coli with both plasmids inside will be co-cultured with another strain of E. coli with a plasmid with chloramphenicol resistance. The cultures will be grown together overnight and then plated on LB plates with a mix of the antibiotic resistance to see if any of the different plasmids have transferred over. The number of colonies from this experiment will be compared to the same experiment, but with pAWP78 and pCM80 with no inserts as a baseline.