Revision as of 15:38, 4 June 2012 by Lisa.O (Talk | contribs)

Week 1 (May 1-4)

This was the first week where we met with other team members and summarized the primary subprojects the team will be tackling this coming summer.

Week 2 (May 7-11)

During this week literature search was performed.

Week 3 (May 14-18)

During this week literature search was performed.

Week 4 (May 22-25)

During this week, strains of Pseudomonas fluorescens PF-5 were obtained. Two cultures were started by adding 500 µL stock to 10 mL LB media containing 50 mg/L ACROS Napthenic Acids. These cultures were grown at 30°C overnight, shaking at 110 rpm.

Overnight cultures were then streaked on LB agar the following day with various types and concentrations of antibiotics in order to determine the susceptibility profile of the organism. This was necessary in order to determine what marker could be used on a transposon to allow for selection of organisms with sucessful transposon insertions. These plates were grown overnight to look for death or growth, and the following results were obtained;

Gentamycin Kanamycin Chloramphenicol Tetracycline
25 µg/ml = no growth 5 µg/ml = slight growth 5 µg/ml = growth 50 µg/ml = no growth
50 µg/ml = no growth 10 µg/ml = slight growth 10 µg/ml = growth 100 µg/ml = no growth
100 µg/ml = no growth 25 µg/ml = no growth 25 µg/ml = growth 200 µg/ml = no growth
50 µg/ml = no growth 50 µg/ml = growth

Based on these results, it was determined that Kanamycin, Gentamycin, and Tetracycline could be used as the selectable marker on the transposon, while Chloramphenicol could not as the strain is naturally resistant. Glycerol stocks of the strains were also made at this time from a fresh overnight culture.

Week 5 (May 28-June 1)

At the beginning of this week we spoke to Dr. Michael Hynes, who was able to give us E. coli SM10 and SM17-1 cells containing the plasmid pOT182. This plasmid contains an E. coli origin of replication, allowing it to act as a suicide vector when transferred to a different bacterial species. pOT182 contains a Tn5 transposon element containing a promotorless lacZ gene, genes for tetracycline resistance as well as a beta-lactamase, and also transposase and an E. coli origin of replication. These elements are bordered by insertion element sequences which are recognised by the transposase. When transferred to a different host through conjugation, the plasmid itself can no longer replicate. The transposase however can recognise and transfer the sequence between the insertion elements in a cut-and-paste fashion randomly into the genome. In this fashion, the tetracycline and beta-lactam resistant traits would only persist in cells in which the transposon has jumped into the genome, allowing these antibiotics to select for transposon positive cells. The lacZ protein will only be produced if the transposon jumps in frame downstream of a promotor, and in this case would allow for a lacZ based assay of the promotors response. The E. coli origin of replication present in the transposon allows for the self-cloning of the transposon in a plasmid format after genomic digestion and circularization and transformation into E. coli, allowing for the sequence of bordering gene fragments to be determined easily and therefore mapping the transposon in the genome.

Cultures of E. coli SM10 and P. fluorescens PF-5 were grown up overnight in shakers at 37°C and 30°C respectively. SM10 was grown in LB + 10 µg/ml Tet, and PF-5 was grown in LB + 50 mg/L ACROS napthenic acids. In the morning, the SM10 culture was subcultured (1/4) into LB without antibiotics and allowed to grow for an additional 4h. After this, 3 replicates of mating mixtures were made with 500 µl of each culture were mixed, and the cells were spun down and resuspended in 50 µl of LB. These samples were then plated in separate spots on LB agar. Additional spots were made in the same fashion with just PF-5 culture and just SM10 culture as controls, and the plate was incubated at 37°C overnight. In the morning, each spot was resuspended in 500µl sterile water + 25 µg/ml Tet, and dilutions of 1, 1/10, 1/100, and 1/1000 of each mating mixture was plated on Pseudomonas Isolation Agar (PIA) + 10µg/l Tet + 50 mg/L ACROS napthenic acids. These cultures were allowed to grow at 30°C over the weekend.

The purpose of the PIA is to selectively allow the growth of the PF-5 strain, while killing off the donor SM10 strain. The tetracycline is designed to select for the positive transposon mutants in the PF-5 strain, as the only way that tetracycline resistance would be acquired (barring spontaneous mutation events) would be if the transposable element had jumped into the genome of the cell. We decided to use 10 µg/ml as the concentration of the tetracycline in the plates because we believed that 50 µg/ml would be too high for even strains carrying resistance to survive. Seeing as 10 µg/ml was effective for E. coli, we chose to try this. After streaking the PF-5 culture on the plate to test for its resistance however, it was found that the strain without the transposon was able to grow slightly on the plates, meaning that the concentration of antibiotic is not high enough to properly select for transposon mutants v.s. untransformed cells. In order to try to remedy this, the mating spots were resuspended in a mixture containing a higher dose of antibiotics. The results of this experiment are pending.

Week 6 (June 4-8)