Phage Conjugation Lab Report

1. The overall goal of this lab was to carry out experiments that clearly demonstrate the different ways DNA genetic information, specifically transduction and conjugation. The first half of this experiment focused on exploring the mechanisms of transduction. This was done by creating a spot titer plate for phage carrying kanamycin resistance and E. coli. E. coli was then proven to have gained kanamycin resistance throughout transduction as demonstrated by its ability to grow on a medium containing kanamycin. The second half of this experiment focused on exploring the concept of conjugation. Our experiment specifically focused on conjugation of resistance genes to two antibiotics nalidixic acid (NalR) and chloramphenicol (CamR). Conjugation
A lambda temperate phage was used meaning it is capable of being maintained in the lysogenic phase. However, depending on the temperature the phage my become lytic. The cl repressor gene was modified to allow for optimum development of the virus. The phage was initially started in the lysogenic phase at 30 degrees celsius. The temperature was then shifted to 42 degrees celsius which caused the phage to no longer be induced and for the repressor to misfold. Then shifting the temperature to 37 degrees celsius caused the phage to enter optimum outgrowth. Chloroform was added after one hour to lyse any living cells and lyse any remaining phages. The phage supernatant mixture was then serially diluted by a 100 μL factor to 10^-5, 10^-6, 10^-7 and 10^-8. The dilutions were then spread on a medium to infect a lawn of bacteria, or E. coli. 25 μL spot titers were used on each quadrant. Lysis of phages were observed over the next 24 hours. Lysis of phages is indicated by the clear/turbid plaques shown in the photo. The clear plaques are the results of replication and lysis of the bacteriophage. A turbid plaques indicates that the phage is not fully lysed yet. No turbidity was observed in the
It is known that NaIR is tryptophan (+) or has the ability to synthesize tryptophan if needed. Additionally it is also known that CamR is tryptophan (-) and does not have the ability to synthesize tryptophan. The ability or genes needed to synthesize tryptophan are not transferred in through conjugation =, presumably because such genes are located on the chromosome. Therefore, by plating the transconjugant on a tryptophan lacking medium one can discern the direction of transfer by growth or no growth of the transjoncjaget. It was shown that only B, B’, T, and T’ displayed growth on the tryptophan (-) medium. This thus proves that NaIR was the recipient and CamR was the donor. If CamR (trp -) had been the donor, T and T’ woud have displayed no growth. Furthermore, it may assumed this direction of transfer is the result of the NaIR gene being located on a chromosome and CamR being located on a