Part:BBa_K640005:Experience
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Applications of BBa_K640005
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Comparison between BBa_K640004 and BBa_K640005
As we mentioned before, there is an existing oriT in the registry (BBa_J01003), that the 2008 Heidelberg team had previously characterized in 2008 for the conjugation of plasmids in E. coli. This is the OriT that is present in our BBa_K640006 construct, which contains the oriT and our ori1600 (BBa_K640003). We had hypothesized that our OriT, given its longer sequence, may contain elements that improve the efficiency of conjugation to Pseudomonas spp. To test this, we did an assay using E. coli conatining our BBa_K640004 part (which contains our oriT: BBa_K640002 and BBa_K640003) as well as E. coli containing BBa_K640006.
We used a simpler conjugation assay, adapted from Serna et al., 2010. Overnight cultures of the donor and the recipient were prepared in LB medium. Aliquots of 500 μl were mixed to obtain a donor/recipient ratio of 1:1. Each mixture was centrifuged to pellet the cells, and the supernatant was discarded. The cells were resuspended in 50 μl of LB broth. Mating mixtures were incubated at 37° for 4 h. Diluted and undiluted aliquots were then spread on selective plates.
Results
The image below shows our results. On the top, E. coli cells containing the BBa_K640004 construct (which contains our oriT) were used. On the bottom row, on either end, we see plates where E. coli cells containing a kanamycin resistant plasmid, but no OriT or ori1600 construct were used. These are pink in color, with no cells growing,indicating that Pseudomonas and E. coli are not able to naturally conjugate plasmids without our part. In the middle of the bottom row, we see plates where E. coli cells containing the BBa_K640005 construct was used (where we use the registry oriT with our ori1600). As we can see, the plates with the BBa_K640004 are much lighter in color, than the plates where the BBa_K640005 construct was used. This is indicative that there is increased pseudomonas growth, when we use our oriT, as compared to the registry oriT. This was exactly what we had expected, and we hope to have more quantitative data in the next few weeks!