Team:Calgary/Notebook/Transposon

From 2012.igem.org

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<h2>Week 1 (May 1-4)</h2>
<h2>Week 1 (May 1-4)</h2>
<p>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.</p>
<p>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.</p>
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<h2>Week 2 (May 7-11)</h2>
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<p> During this week literature search was performed.</p>
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<h2>Week 3 (May 14-18)</h2>
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<p> During this week literature search was performed.</p>
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<h2>Week 4 (May 22-25)</h2>
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<p> During this week, strains of <i> Pseudomonas fluorescens</i> PF-5 were obtained. Two cultures were started by adding 500 &micro;L stock to 10 mL LB media containing 50 mg/L ACROS Napthenic Acids. These cultures were grown at 30&deg;C overnight, shaking at 110 rpm.</p>
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<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>
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<p><center><b>Gentamycin</b></p>
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<p>25 &micro;g/ml = no growth</p>
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<p>50 &micro;g/ml = no growth</p>
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<p>100 &micro;g/ml = no growth</p>
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<p>
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<p><b>Kanamycin</b></p>
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<p>5 &micro;g/ml = slight growth</p>
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<p>10 &micro;g/ml = slight growth</p>
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<p>25 &micro;g/ml = no growth</p>
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<p>50 &micro;g/ml = no growth</p>
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<p>
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<p><b>Chloramphenicol</b></p>
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<p>5 &micro;g/ml = growth</p>
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<p>10 &micro;g/ml = growth</p>
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<p>25 &micro;g/ml = growth</p>
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<p>50 &micro;g/ml = growth</p>
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<p>
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<p><b>Tetracycline</b></p>
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<p>50 &micro;g/ml = no growth</p>
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<p>100 &micro;g/ml = no growth</p>
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<p>200 &micro;g/ml = no growth</center></p>
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<p>
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<p>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.</p>
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<h2>Week 5 (May 28-June 1)</h2>

Revision as of 22:33, 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;

Gentamycin

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.

Week 5 (May 28-June 1)