Team:UANL Mty-Mexico/Safety/stability test

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<h2>Safety</h2>
<h3>Stability Test</h3>
<h3>Stability Test</h3>
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<p>In the case of an accidental release (or even an intentional) of our plasmids, we'd be interested in knowing for how long will our cells keep the DNA with put on them, that is, how stable a plasmid is in the absence of a selective pressure, such as the antibiotic commonly used to specifically grow transformed cells. Plasmid stability tests are commonly employed to determine for how many generations can a cell culture keep a foreign plasmid in non-selective conditions.</p>
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<p>We started with the assumption that our constructions -RNATs and reporter proteins- do not confer a selective advantage to the cells. In other words, we assumed that the replication origin of each plasmid should be the main factor that affects plasmid stability.</p>
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<p>The core plasmids we needed to test were pUC and pSB, since all of our constructions are based on this skeletons.</p>
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<p>We followed the protocol found in <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013244" target="1">Bryksin and Matsumura, 2010</a>,  with a 100 mL culture that was re-inoculated with 100 &mu;L of the culture of the previous day.</p>
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<center><h2>pUC 57</h2></center>
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                 text: 'Plasmid Stability',
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                 text: 'Plasmid Stability pUC57',
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<p>We observed that after day 3 (generation 30), pUC showed a pronounced decrease on plasmid retention (from 30% to 1%) and reached 0% on day 5.</p>
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<p>This allows us to conclude that, in the case of a release to the environment, when the retention of the plasmid is left to the effect of genetic drift, at least 30 generations will pass until almost no cell retains the plasmid pUC and its derivatives that harbor genes that do not confer a selective advantage.</p>
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<center><h2>pSB 1C3</h2></center>
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            subtitle: {
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                text: 'Population percentage retaining antibiotic resistance',
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            xAxis: {
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                categories: ['Day 1', 'Day 2', 'Day 3', 'Day 4', 'Day 5','Day 6','Day 7','Day 8']
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<p>We observed that after day 8 (generation 80), pSB showed a decrease on plasmid retention (from up to 100% to 2%) and is suposed to reach 0% on day 10.</p>
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<p>In the case of a release to the environment, at least 90 generations will pass until almost no cell retains the plasmid pSB and its derivatives that harbor genes that do not confer a selective advantage.</p>
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<b>Protocol</b>
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<h3>Protocol</h3>
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<p>Plasmid stability test in E. coli:</p>
<p>Plasmid stability test in E. coli:</p>
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<p>A. Inoculate a flask with 10 ml of LB medium without antibiotic.</p>
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<ol><li>Inoculate a flask with 10 ml of LB medium with the proper antibiotic.</li>
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<p>B. Grow overnight until saturation is reached.</p>
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<li>Grow overnight until saturation is reached.</li>
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<p>C. Make serial dilutions (1:6, 1:7, 1:8).</p>
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<li>Inoculate 100 mL of fresh LB without antibiotic with 100 &mu;L of the overnight culture. Simultaneosuly, inocculate a dilution series (from 10<sup>-4</sup> to 10<sup>-8</sup>) of the overnight culture on two series of plaques, one with the antibiotic and one without.</li>
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<p>D. Inoculate in LB medium without antibiotic and incubate for 80 Generations.</p>
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<li>Repeat the procedure for eight days (approximately 80 generations, according to <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013244" target="1">Bryksin and Matsumura, 2010</a>.</li>
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<p>E. Inoculate each generation in petri dishes with LB medium with and without antibiotic, incubate overnight.</p>
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<li>Calculate each day the percentage of Colony Forming Units (CFUs) that retain the plasmid by dividing the number of cells growing in plaques with antibiotic by the number of cells growing without antibiotic.</li></ol></p>
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<p>F. Count colonies and calculate the percentage of plasmid retention.</p>
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<center><img src="https://static.igem.org/mediawiki/2013/d/d6/StabilityProtocolthermocoli2013.jpg" data="holder.js/600x300/auto" alt="Generic placeholder image" width="600px" height="400px"class="img-rounded"></center>
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<center><p><h3>Overview of protocol</h3></p></center>
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Safety

Stability Test

In the case of an accidental release (or even an intentional) of our plasmids, we'd be interested in knowing for how long will our cells keep the DNA with put on them, that is, how stable a plasmid is in the absence of a selective pressure, such as the antibiotic commonly used to specifically grow transformed cells. Plasmid stability tests are commonly employed to determine for how many generations can a cell culture keep a foreign plasmid in non-selective conditions.

We started with the assumption that our constructions -RNATs and reporter proteins- do not confer a selective advantage to the cells. In other words, we assumed that the replication origin of each plasmid should be the main factor that affects plasmid stability.

The core plasmids we needed to test were pUC and pSB, since all of our constructions are based on this skeletons.

We followed the protocol found in Bryksin and Matsumura, 2010, with a 100 mL culture that was re-inoculated with 100 μL of the culture of the previous day.


pUC 57


We observed that after day 3 (generation 30), pUC showed a pronounced decrease on plasmid retention (from 30% to 1%) and reached 0% on day 5.

This allows us to conclude that, in the case of a release to the environment, when the retention of the plasmid is left to the effect of genetic drift, at least 30 generations will pass until almost no cell retains the plasmid pUC and its derivatives that harbor genes that do not confer a selective advantage.


pSB 1C3


We observed that after day 8 (generation 80), pSB showed a decrease on plasmid retention (from up to 100% to 2%) and is suposed to reach 0% on day 10.

In the case of a release to the environment, at least 90 generations will pass until almost no cell retains the plasmid pSB and its derivatives that harbor genes that do not confer a selective advantage.

Protocol

Plasmid stability test in E. coli:

  1. Inoculate a flask with 10 ml of LB medium with the proper antibiotic.
  2. Grow overnight until saturation is reached.
  3. Inoculate 100 mL of fresh LB without antibiotic with 100 μL of the overnight culture. Simultaneosuly, inocculate a dilution series (from 10-4 to 10-8) of the overnight culture on two series of plaques, one with the antibiotic and one without.
  4. Repeat the procedure for eight days (approximately 80 generations, according to Bryksin and Matsumura, 2010.
  5. Calculate each day the percentage of Colony Forming Units (CFUs) that retain the plasmid by dividing the number of cells growing in plaques with antibiotic by the number of cells growing without antibiotic.

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Overview of protocol

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