Team:Calgary/Notebook/PromoterScreen

From 2012.igem.org

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Revision as of 22:16, 29 May 2012

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.

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 <td><b>Gentamycin</b></td>
 <td><b>Kanamycin</b></td>