Week 7 - Growing Bacteria

Week seven of my internship included a lot of reading papers and waiting for bacteria to grow.  This week and next, we are working on conjugating the plasmid containing the delta DNA fragment (the one without the gene of interest) into Teredinibacter turnerae.  However, this step requires aligning the growth of the E. coli and the T. turnerae before conjugation can start.  As a result, the process must be timed carefully in several short steps over several days.  Hence the reading and waiting.  Overall, though, I learned quite a bit.  The extra time allowed me to research more in to some of the topics we are covering, and I got another chance to work with the Geneous genetics program.

Monday I performed the colony PCR check on the E. coli from last week which hopefully contain the Δ2288 fragment.  All ten of the first colonies showed positive results for presence of the fragment, so I chose three to inoculate.  At the same time, I inoculated a beaker with T. turnerae for use in conjugation with the S17 E. coli from last week which contain the Δ1990 fragment.  I also re streaked all 16 of the Δ2288 colonies onto an LB/Ampicillin plate to check for the presence of the pGEMe vector that we used earlier on which should have been removed during the digestion process last week.  Later in the afternoon, Hiroaki showed me how to set up the conjugation process to transfer the fragment-containing-plasmid from the E. coli to the Teredinibacter turnerae.

On Tuesday I extracted the Δ2288/pDM4 DNA plasmid from the DH5 E. coli that I inoculated yesterday afternoon.  Then I transformed it into another strain of E. coli, S17 λpir, which can perform conjugation (necessary to get the plasmid into the T. turnerae bacteria), and plated these onto an LB/Kanamycin agar plate.  I also inoculated the S17 λpir bacteria from last week containing the Δ1990 fragment. 

Wednesday I didn’t have a whole lot to do, so I spent quite a bit of time reading journal articles.  In the morning I removed the plates containing S17 E. coli from the incubator, wrapped them in parafilm and put them in the 4°C refrigerator.  Both plates grew well, so next week I will start the inoculation process of T. turnerae and these cells so that I can set up a conjugation process in the middle of next week.  I also set up the conjugation process for the Δ1990 fragment.  This took a while, because I had to save two stock solutions in glycerol of each set of cells which were stored in two separate -80°C freezers.  In addition, the process to rinse and combine the cells requires a lot of centrifugation. 

Thursday morning I re-streaked the conjugation mixture onto SMB-N-Sigma/Km 50 plates.  These plates contain cellulose as their sole carbon source, allowing us to select for only T. turnerae cells, because E. coli cannot metabolize cellulose.  The presence of antibiotics allows us to select for only cells which have successfully integrated the entire Δ1990/pDM4 plasmid into their DNA.  Later, I used the Geneous program to create primers for a gene in a new region which Hiroaki is hoping to work on soon.  The process was a lot more involved than I had thought it would be, but it was really interesting.