Team:MichiganState/Results/HGT

Results

General Anoxic HGT Biosensor Bioreactor Design

HGT Results

Primary Goal

The initial goal of this project was to limit the transfer of plasmid DNA between different organisms using a toxin-antitoxin system. The toxin was designed to be placed in the pCM80 plasmid, where a blue-white screening could be performed, and the antitoxin was designed to be placed in the pAWP78 plasmid. Then once they were transformed they would be placed into E. Cloni 10G commercially competent cells to test the effectiveness of the inserts.

Figure 1a (left) pCM80 construct with the ghoT toxin inserted into the LacZ gene that allows for a blue-white screening to determine successful transformation. Figure 1b (right) pAWP78 construct with ghoS antitoxin inserted.

Confirming the Antitoxin Transformation

After PCRs were run and Gibson assembly was performed the products were transformed into chemically competent E. cloni 10G cells for expression. To confirm that we had the desired inserts we had a colony PCR was run on the transformant colonies.

Figure 2 A colony PCR gel for the ghoS antitoxin insert in pAWP78. The gel shows the inserts around 750 bp, which is the correct size for the ghoS gene.

Testing the Toxin

The toxin in pCM80 was inserted alone into E. cloni 10G and was tested with different concentrations of cumate, which would induce the production of the toxin. The toxin was tested on a plate reader with 5 replicates of each concentration with colonies suspected to have the insert and 3 replicates of cells transformed with pCM80 no insert. The plate reader then ran for 12 hours to see if the bacteria could grow when the toxin was being expressed.

Figure 3 This graph shows the growth curve, measured by OD600 over time, of the E. cloni 10G strain with the pCM80+ghoT plasmid (solid lines) and the E. cloni 10G with only the pCM80 plasmid (dotted lines). The different colors represent varying amounts of cumate added (concentrations in the legend) because the ghoT promoter is cumate inducible. As cumate concentration is increased the strains with the toxin gene show significantly less growth than the strains with the blank plasmid.

Testing the Toxin and antitoxin together

After a successful test to prove that the antitoxin functioned as expected E. cloni 10G was transformed with both the toxin and the antitoxin plasmids to see if the antitoxin functioned as well. The transformants were then run on the plate reader the same as the toxin was. The results of this assay, however, showed that the antitoxin either did not keep up with the toxin or that the antitoxin did not function at all.

Future Work

In the future more work would have to be done to get a functioning antitoxin, such as replacing the ghoS promoter with a stronger one to see if the amount of antitoxin was the problem. After the antitoxin works then tests could be performed to see if this system limits plasmid transfer.