Difference between revisions of "Team:Austin UTexas/Results"

Conjugation

We have attempted to conjugate GFP into both G. oxydans and G. hansenii with a Diaminopimelic Acid (DAP) auxotrophic strain of E. coli . The plasmid contains the vector pMMB67EH, the promoter PA-1, GFP and a spectinomycin resistance gene.

The first conjugation was done with KOM strains 4 (G. oxydans) 5 ( G. oxydans and 15 ( L. fermentati ). We attempted these conjugations before sequencing the recipient strains, so that is why we tried to conjugate into L. fermentati . First, a mixture between a KOM strain and the DAP auxotroph strain were plated on a LB+DAP solid medium to allow for conjugation to occur. After 24 hours of incubation, I scraped up the growth and plated each conjugation mixture onto a LB+Spec plate.

LB+SPEC plates that contain conjugation mixtures of KOM 4, 5 and 15 ( L. fermentati )

Next, we viewed the potential transconjugants on a fluorescence microscope.

Using a fluorescent microscope, this was a picture taken of the G. oxydans strain, KOM 4, without the plasmid that contains GFP.

Using a fluorescent microscope, this was a picture taken of the potential transconjugant, KOM 4, with the plasmid GFP. 16s sequencing was still needed to confirm successful conjugation.

We then picked these glowing colonies and then after streaking them out onto more LB+Spec plates, we attempted to use 16s sequencing to confirm successful conjugation. After troubleshooting our 16s procedure, we were finally able to obtain a viable sequencing result. However, all of the glowing colonies were identified as E. coli. For the next round of conjugation, we used a strain of both G. oxydans and Gluconacetobacter hansenii from the American Type Culture Collection (ATCC).

This is a LB+DAP plate on a dark reader that has four different conjugations occurring at one time. The two left quadrants have the same ATCC strain of G. oxydans,, while the two quadrants on the right have Ga. hansenii.

These growths were then scraped up and plated onto a LB+Spec plate.

These are my potential transconjugants on a LB+DAP plates. The dark reader was used when taking this picture. The top two are G. oxydans while the bottom two are G. hansenii.

We then picked isolated colonies and streaked them out onto LB+DAP plates. After using 16s sequencing on the potential transconjugants, we encountered an anomaly. Instead of amplifying the 16s gene, we recieved the sequence of the L,D Transpeptidase gene of E. coli . We plan on repeating the 16s procedure.