Team:UANL Mty-Mexico/Safety/stability test

From 2013.igem.org

(Difference between revisions)
 
(29 intermediate revisions not shown)
Line 2: Line 2:
<html>
<html>
 +
 +
<h2>Safety</h2>
<h3>Stability Test</h3>
<h3>Stability Test</h3>
-
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.8.2/jquery.min.js"></script>
+
<div class="justified">
 +
<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>
 +
<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>
 +
<p>The core plasmids we needed to test were pUC and pSB, since all of our constructions are based on this skeletons.</p>
 +
<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>
-
<div id="container" style="min-width: 310px; height: 400px; margin: 0 auto"></div>
+
<br>
 +
<center><h2>pUC 57</h2></center>
 +
<!--chart container (charts will appear where the container is put--><div id="chart-A" class="chart"></div> <!-- End container for Chart A -->
 +
<!--Interactive javascript charts (highcharts.com licensed under Creative Commons Attribution-NonCommercial 3.0 License)-->
 +
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.8.2/jquery.min.js"></script>
 +
<!--begin pUC57 retention chart script-->
<script type="text/javascript">
<script type="text/javascript">
$(function () {
$(function () {
-
         $('#container').highcharts({
+
         $('#chart-A').highcharts({
             title: {
             title: {
-
                 text: 'Plasmid Stability',
+
                 text: 'Plasmid Stability pUC57',
                 x: -20 //center
                 x: -20 //center
             },
             },
Line 48: Line 59:
     });
     });
   </script>
   </script>
 +
<!-- end pUC57 chart script-->
 +
<br>
 +
<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>
 +
 +
<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>
 +
<br>
 +
<center><h2>pSB 1C3</h2></center>
 +
<!--second chart container--><div id="chart-B" class="chart"></div> <!-- End container for Chart B -->
 +
<!--begin pSb retention chart script-->
 +
<script type="text/javascript">
 +
$(function () {
 +
        $('#chart-B').highcharts({
 +
            title: {
 +
                text: 'Plasmid Stability pSB1C3',
 +
                x: -20 //center
 +
            },
 +
            subtitle: {
 +
                text: 'Population percentage retaining antibiotic resistance',
 +
                x: -20
 +
            },
 +
            xAxis: {
 +
                categories: ['Day 1', 'Day 2', 'Day 3', 'Day 4', 'Day 5','Day 6','Day 7','Day 8']
 +
            },
 +
            yAxis: {
 +
                title: {
 +
                    text: 'Plasmid retention'
 +
                },
 +
                plotLines: [{
 +
                    value: 0,
 +
                    width: 1,
 +
                    color: '#808080'
 +
                }]
 +
            },
 +
            tooltip: {
 +
                valueSuffix: '%'
 +
            },
 +
            legend: {
 +
                layout: 'vertical',
 +
                align: 'right',
 +
                verticalAlign: 'middle',
 +
                borderWidth: 0
 +
            },
 +
            series: [{
 +
                name: 'pSB1C3',
 +
                data: [113, 96, 96, 82, 92, 49,60,2]
 +
               
 +
            } ]
 +
        });
 +
    });
 +
  </script>
 +
<!-- End psb retention chart script -->
 +
<br>
 +
<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>
 +
<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>
<script src="http://code.highcharts.com/highcharts.js"></script>
<script src="http://code.highcharts.com/highcharts.js"></script>
<script src="http://code.highcharts.com/modules/exporting.js"></script>
<script src="http://code.highcharts.com/modules/exporting.js"></script>
-
<br><br>
 
-
<div class="justified">
+
<h3>Protocol</h3>
-
<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 abscence 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>
+
-
<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>
+
-
<p>The core plasmids we needed to test were pUC and pSB, since all of our constructions are based on this skeletons. However, due to time constrains, we were only able to test pUC.</p>
+
-
<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>
+
-
<b>Protocol</b>
 
-
<hr>
 
<p>Plasmid stability test in E. coli:</p>
<p>Plasmid stability test in E. coli:</p>
<ol><li>Inoculate a flask with 10 ml of LB medium with the proper antibiotic.</li>
<ol><li>Inoculate a flask with 10 ml of LB medium with the proper antibiotic.</li>
<li>Grow overnight until saturation is reached.</li>
<li>Grow overnight until saturation is reached.</li>
<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>
<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>
-
<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>). Nevertheless, we stopped the experiment when the culture reached 0% retention.</li>
+
<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>
-
<li>Calculate each day the percentage of Colony Forming Units (CFUs) that retain the plasmid by divinding the number of cells growing in plaques with antibiotic by the number of cells growing without antibiotic.</li></ol></p>
+
<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>
-
<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>
 
-
<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>
+
<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>
 +
<center><p><h3>Overview of protocol</h3></p></center>
 +
 
-
<p>Future work will be done to determine the stability of plasmid pSB and to let both plasmids grow after the day they reach 0% retention to see if the plasmid somehow re-emerges.</p>
 
<!-- Site footer -->
<!-- Site footer -->

Latest revision as of 01:56, 29 October 2013

Carousel Template for Bootstrap


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.

Generic placeholder image

Overview of protocol

Creative Commons License