"
Team:Calgary/Notebook/PromoterScreen
From 2012.igem.org
| Line 16: | Line 16: | ||
<h2>Week 4 (May 22-25)</h2> | <h2>Week 4 (May 22-25)</h2> | ||
| - | <p> During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 | + | <p> During this week, strains of <i> Pseudomonas fluorescens</i> 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.</p> |
<p> Overnight cultures were then plated on LB agar the following day on various types and concentrations of antibiotics in order to determine the susceptibility profile. This was necessary in order to determine what selectable marker could be used on a transposon. These plates were grown overnight, and the following results were obtained; </p> | <p> Overnight cultures were then plated on LB agar the following day on various types and concentrations of antibiotics in order to determine the susceptibility profile. This was necessary in order to determine what selectable marker could be used on a transposon. These plates were grown overnight, and the following results were obtained; </p> | ||
<p><center><b>Gentamycin</b></p> | <p><center><b>Gentamycin</b></p> | ||
Revision as of 22:32, 28 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 plated on LB agar the following day on various types and concentrations of antibiotics in order to determine the susceptibility profile. This was necessary in order to determine what selectable marker could be used on a transposon. These plates were grown overnight, and the following results were obtained;
25 µg/ml = no growth
50 µg/ml = no growth
100 µg/ml = no growth
Kanamycin
5 µg/ml = slight growth
10 µg/ml = slight growth
25 µg/ml = no growth
50 µg/ml = no growth
Chloramphenicol
5 µg/ml = growth
10 µg/ml = growth
25 µg/ml = growth
50 µg/ml = growth
Tetracycline
50 µg/ml = no growth
100 µg/ml = no growth
200 µg/ml = no 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.
