In the time since our last blog post we have tried several attempts to perform a successful conjugation with multiple strains of Pseudoalteromonas and Planococcus associated with the accessory nidamental gland of the squid. We follow the conjugation protocol for the conjugation. Conjugation is how bacteria exchange genetic information. In this case our E. coli conjugates contain a transposon, which basically randomly inserts into DNA and we are hoping for this transposon to insert in the codon that codes for biofilms for our bacterial samples. Here are the basic steps from this protocol: first overnight cultures of or samples are grown. The next day we combine our intended recipient bacteria with the E. coli conjugant that has the transposon and pellet them using a centrifuge. We then resuspend the pellet and then spot the mixture of the two on a plate. We then let the bacteria grow over night at room temperature where the two should conjugate. The next day we scrape the spot off the plate, resuspend in Luria broth and pipette 10-50 microliters onto fresh plates and spread it evenly using sterile glass beads. These plates are then grown at room temperature overnight.
There is a selective antibiotic marker for erythromycin that is combine into the recipient bacterium during the transposition. This selective agent allows for us to grow up our transformed bacterial colonies. What we are hoping to get for results include interruption of the genes for biofilm and pigment formation. We will be screening for colonies with no biofilms and impaired pigment formation. However we have had difficulties with the bacterial conjugation and we have not had high numbers of bacterial transformants. Which means for some reason our bacteria isn’t conjugating with E. coli and we are trouble shooting this process.
We have recently ordered fresh antibiotic to add to the plates which should limit the growth of the donor E. coli but allow for our transformed bacteria to grow. One other factor that can be optimized to increase transformation is increasing the moisture content of the media. If this next round is not successful we may need to reorder our conjugate strains.