Difference between revisions of "Team:Exeter/Proof"

Latest revision as of 14:46, 19 October 2016

Proof of Concept

Continuous culture

The main focus of Project: Exepire in the lab was the robustness of kill switches in real world conditions. By looking at the effectiveness of the kill switches in continuous culture we have begun to show potential failure rates over time. By simulating a continuous culture that would take place on a much larger scale in industry, we have shown the potential failures that need to be addressed if kill switches are to replace traditional chemical and physical bio-containment.

Before starting the project we spoke to Prof. Robert Beardmore EPSRC Leadership Fellow in the Mathematical Biosciences at Exeter University. Much of his research has been into antibiotic resistance. We discussed how high selection pressure is applied by prolonged use of antibiotics and how kill switches may be analogous to this. It is clear that cells which develop a mutation that inactivates the kill switch would be strongly selected for. It was estimated that functional loss of the kill switch would occur in a short amount of time as a result, and if this was the case, could have strong implications for kill switch longevity. To test this we decided to use a ministat to perform a continuous culture. The ministat was developed in the Dunham lab at the University of Washington (Miller et al, 2013). Each ministat chamber is fed from its own media container via a peristaltic pump, with the culture volume set by the height of the effluent needle in the chamber. Air is bubbled through flasks of water to hydrate it and then used to agitate the culture. Chambers were inoculated with freshly transformed E. coli BL21 (DE3) and samples taken to test if the kill switches were still viable. By simulating in miniature how a kill switch might behave in an industrial setting, the ministat provides a proof of concept for how a kill switch might be maintained in larger chemostats during a continuous culture. A protocol for running experiments in the ministat can be found here

Media container used to feed a single ministat chamber.

Peristaltic pump

Ministat chambers in heatblock and 1 litre Duran bottle used to collect effluent

Ministat running a preliminary experiment to calibrate parameters such as dilution rate and temperature of the heat block. 50 ml burettes used here to accurately measure effluent levels. 1 litre Duran bottles were used for effluent collection in the main experiment due to greater volumes of effluent.

Our own growth curve was performed to determine the maximum specific growth rate of E. coli BL21 (DE3) in our lab, but could not be conducted for a sufficient length of time to be accurate. A maximum specific growth rate value of 1.730 was used (Cox, 2004). The ministat must be set to a flow rate at which dilution rate is less than maximum specific growth rate. This prevents the culture being washed out of the growth chambers. The dilution rate of the culture was calculated by measuring flow rate at a setting of 7.5 rpm on the peristaltic pump. For practical reasons the pump could not be run faster than this due to the amount of media needed. The dilution rate was set at 0.2 which produced cultures that grew at an average OD of 3.47 for KillerRed samples, 3.64 for KillerOrange samples and 3.17 for lysozyme samples. The ministat must be set to flow rate at which dilution rate is below the maximum specific growth rate. This prevents the culture being washed out of the chamber. OD was measured daily with a Bug Lab OD scanner. When the same sample was measured in a tecan infinite 200 pro plate reader the Bug Lab showed reading approximately three times higher. The difference between the samples was consistent regardless of the method used to measure OD.

Ministat experiment

All samples from the ministat were tested using the KillerRed, KillerOrange protocol found here. Glycerol stocks were made from the samples taken at each time interval, testing was done using these glycerol stocks.

The following graphs show the average number of colonies of samples taken at 0 h, 24 h, 120 h and 168 h of continuous culture and then tested in the light box. Values were averaged across three biological repeats. Colonies were not counted above 300 and so this is the maximum value given. All samples were induced to a final concentration of 0.2 nM IPTG. All samples were diluted 1000 times in a final volume of 4.5 ml liquid broth (LB). Error bars represent the standard error of the mean.

Fig. 14. Comparison of CFUs formed by KillerRed exposed to light and kept in the dark for each sample taken from the ministat. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs.

Fig. 15. Comparison of CFUs formed by KillerOrange exposed to light and kept in the dark. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs. The effect is not as obvious in KillerOrange compared to KillerRed as the starting efficiency of KillerOrange is lower.

Discussion

The continuous culture of KillerRed showed a 15 fold increase in the percentage of viable cells after 168 hrs. A similar pattern is shown for KillerOrange but with around a two fold increase. Both KillerRed and KillerOrange show greater numbers of colonies forming over time (Fig. 14 & 15). This number approaches the amount produced in the dark condition by 168 hrs. The average fluorescence reading for 0 hr KillerRed samples was 506.3 A.U (recorded at an average OD of of 0.745). After 168 hrs the average fluorescence reading was 436 A.U (at an average OD of 0.96). It seems unlikely due to the readings being similar that a mutation has occurred in the kill switch itself. As fluorescence is proportional to the amount of ROS being produced, up regulation of native E. coli enzymes that mitigate the effects of ROS may be the cause of the increase in cell survival. Future transcriptome analysis could provide interesting data on the mechanism of this change, this was unfortunately beyond the scope of this project. This shows that there may be many ways for bacteria to circumvent the effects of a kill switch given the high selection pressure they pose.

@@ Line 635: / Line 635: @@
 <h6><u>Ministat experiment</u></h6>
 <p id="pp">All samples from the ministat were tested using the KillerRed, KillerOrange protocol found <a href="#KRKOProt">
-here</a>. Glycerol stocks were made of the samples taken at each time interval, testing was done using these glycerol stocks.</p>
+here</a>. Glycerol stocks were made from the samples taken at each time interval, testing was done using these glycerol stocks.</p>
-<p id="pp"><b>Fig.1,2</b> Average number of colonies after 0 h, 24 h, 120 h and 168 h of continuous culture. Values were
+<p id="pp">The following graphs show the average number of colonies of samples taken at 0 h, 24 h, 120 h and 168 h of continuous culture and then tested in the light box. Values were
-averaged across three biological repeats. A max value of 300 colonies is set as any plate with more than 300 colonies was
+averaged across three biological repeats. Colonies were not counted above 300 and so this is the maximum value given. All samples were induced to a final concentration of 0.2 nM IPTG. All samples were
-not counted and assigned the max value. All samples were induced to a final concentration of 0.2 nM IPTG. All samples were
+diluted 1000 times in a final volume of 4.5 ml liquid broth (LB). Error bars represent the standard error of the mean.</p>
-diluted 1000 times in a final volume of 4.5 ml LB. Error bars represent the standard error of the mean</p>
-<p id="pp"> <b>Fig. 3,4</b> Data from Fig.7,8 represented as percentage viable cells over time. 100% viable is given
-when the CFU count for the kill switch condition equaled the control. Error bars represent the standard error of the mean.</p>
 <div class="row">
      <div class="col-xs-6" style="padding:5px 2% 5px 10%;margin:0;">
          <img src="https://static.igem.org/mediawiki/2016/7/78/T--Exeter--KRcont.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 1. Comparison of CFUs formed by KillerRed exposed to light and kept in the dark.</span>
+             <span class="caption">Fig. 14. Comparison of CFUs formed by KillerRed exposed to light and kept in the dark for each sample taken from the ministat. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs.</span>
 		<br>
 		<br>
@@ Line 654: / Line 652: @@
      <div class="col-xs-6" style="padding:5px 10% 5px 2%;margin:0;">
-         <img src="https://static.igem.org/mediawiki/2016/9/9f/T--Exeter--KRDpercent2.jpg"
+         <img src="https://static.igem.org/mediawiki/2016/d/d0/T--Exeter--KOcont.jpg"
 		style="max-width:100%;margin:auto;display:block;">
-             <span class="caption">Fig. 2. Percentage viable cells of KillerRed exposed to light.</span>
+             <span class="caption">Fig. 15. Comparison of CFUs formed by KillerOrange exposed to light and kept in the dark. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs. The effect is not as obvious in KillerOrange compared to KillerRed as the starting efficiency of KillerOrange is lower. </span>
 		<br>
 		<br>
@@ Line 662: / Line 660: @@
 </div>
-<div class="row">
-    <div class="col-xs-6" style="padding:5px 2% 5px 10%;margin:0;">
-        <img src="https://static.igem.org/mediawiki/2016/d/d0/T--Exeter--KOcont.jpg"
-		style="max-width:100%;margin:auto;display:block;">
-            <span class="caption">Fig. 3. Comparison of CFUS of KillerOrange exposed to light and in the dark.</span>
-		<br>
-		<br>
-    </div>
-<div class="row">
+<h6>Discussion</h6>
-    <div class="col-xs-6" style="padding:5px 10% 5px 2%;margin:0;">
-        <img src="https://static.igem.org/mediawiki/2016/0/01/T--Exeter--KOpercent2.jpg"
-		style="max-width:100%;margin:auto;display:block;">
-            <span class="caption">Fig. 4. Percentage viable cells of KillerOrange exposed to light.</span>
-		<br>
-		<br>
-    </div>
-</div>
-</div>
+<p id="pp">The continuous culture of KillerRed showed a 15 fold increase in the percentage of viable cells after 168 hrs. A similar pattern is shown for KillerOrange but with around a two fold increase. Both KillerRed and KillerOrange show greater numbers of colonies forming over time (Fig. 14 & 15). This number approaches the amount produced in the dark condition by 168 hrs.
+The average fluorescence reading for 0 hr KillerRed samples was 506.3 A.U (recorded at an average OD of of 0.745). After 168
+hrs the average fluorescence reading  was 436 A.U (at an average OD of 0.96). It seems unlikely due to the readings being
+similar that a mutation has occurred in the kill switch itself. As fluorescence is proportional to the amount of ROS
+being produced, up regulation of native <i>E. coli</i> enzymes that mitigate the effects of ROS may be the cause of the increase
+in cell survival. Future transcriptome analysis could provide interesting data on the mechanism of this change, this was
+unfortunately beyond the scope of this project. This shows that there may be many ways for bacteria to circumvent the effects of a kill switch given the high selection pressure they pose.</p>
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