Difference between revisions of "Team:Exeter/Project"

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<div>

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<li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Project">Lab Project</a></li>

+

<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Safety">Safety</a></li>

</ul>

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<li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Awards">Awards</a></li>

−

<li>Special pages</li>

+

<li>Special pages</li>

<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Silver">HP Silver</a></li>

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</ul>

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+

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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

+

By simulating in miniature how a kill switch might behave in an industrial setting, the ministat provides a <a href="https://2016.igem.org/Team:Exeter/Proof">proof

−

of concept for how a kill switch might be maintained in larger chemostats during a continuous culture. A protocol

+

of concept</a> 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 <a href="#MinistatProt">here</a>

Line 833:

Line 834:

which, when irradiated with green and blue light respectively, generate reactive oxygen species (ROS). KillerRed has been

shown to effectively kill cells when exposed to green light (540–580 nm) and is much less effective under blue light

−

(460–490 nm) (Bulina et al, 2006). KillerOrange effectively kills cells when exposed to 450-495nm (Sarkisyan 2015), the range that KillerRed does not. ~~The open Beta~~ barrel of the ~~protein~~ is thought to ~~allow the solvent to come into contact~~ with ~~the chromophore, facilitating the release of reactive oxygen species~~ (~~Carpentier~~ et al, 2009)

+

(460–490 nm) (Bulina et al, 2006). KillerOrange effectively kills cells when exposed to 450-495nm (Sarkisyan 2015), the range that KillerRed does not. There is a β-barrel present in both these proteins. A water-filled channel that is in contact with a chromophore area and located at the cap of the said β-barrel is thought to confer these proteins with their phototoxic capabilities (Pletnev S et al, 2009).

Line 844:

Line 845:

</div>

−

The mechanism by which ROS kill cells ~~is not fully~~ understood~~, partly because they react quickly~~

+

The mechanism by which ROS kill cells isn’t completely understood. However ROS have been shown to have various detrimental roles in cells such as the oxidation of thiols, ascorbate and proteins containing (Fe-S)4 clusters as well as reducing various transition metals (Farr and Kogama, 1991). Prolonged exposure and or high levels of ROS triggers apoptosis like mechanisms (Held, 2015). Our metabolic kill switches build on previous iGEM projects which have used the expression of highly phototoxic

−

~~with contaminating metals~~ to ~~form more reactive species that obscure their own role~~ in oxidation ~~damage~~ (Farr and Kogama, 1991). Prolonged exposure and or high levels of ROS triggers apoptosis like mechanisms (Held, 2015). Our metabolic kill switches build on previous iGEM projects which have used the expression of highly phototoxic

+

fluorescent proteins to kill the cells by exposing the culture to light. In 2013, the iGEM team from Carnegie Mellon developed a phage delivery system of the KillerRed gene, which was then expressed in the infected bacteria, killing it on exposure to light. Carnegie Mellon 2014 continued characterisation of KillerRed and its monomeric form Supernova by analysing their photobleaching characteristics. Neither team tested the longevity of the kill switch or provided details on the light intensity that the cultures were exposed to. We aim to quantify the length of time for which the kill switch remains viable and provide absolute values for the intensity of our light source.

Line 854:

<h6>Method</h6>

−

The following samples were tested for phototoxicity by exposing them to 1.2 µW/cm2 of white light from a 4x8 LED array (Fig. 6) for 6 hrs. Samples were then spread plated and colony forming units (CFUs) were counted. All parts were carried on the pSB1C3 plasmid and transformed into E. coli BL21 (DE3). Samples that were induced were done so with Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 nM.

+

The following samples were tested for phototoxicity by exposing them to 1.2 mW/cm2 of white light from a 4x8 LED array (Fig. 6) for 6 hrs. Samples were then spread plated and colony forming units (CFUs) were counted. All parts were carried on the pSB1C3 plasmid and transformed into E. coli BL21 (DE3). Samples that were induced were done so with Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 nM.

Henceforth samples will be refered to as:

<style>

Line 932:

</div>

−

KillerRed is excited by green/yellow light (540-580 nm) and KillerOrange by blue light (460-490 nm). We constructed a box around the LED array to prevent ambient light entering. Access to the inside of the box was gained through an opening cut in the front. With help from <a href="#">Ryan Edginton</a>, we used a portable spectrometer (Ocean Optics USB2000+VIS-NIR-ES, connected to a CC3 cosine corrector with a 3.9 mm collection diameter attached to a 0.55 mm diameter optical fibre) to measure light spectra and absolute intensity in the visible range.

+

KillerRed is excited by green/yellow light (540-580 nm) and KillerOrange by blue light (460-490 nm). We constructed a box around the LED array to prevent ambient light entering. Access to the inside of the box was gained through an opening cut in the front. With help from <a href="http://emps.exeter.ac.uk/physics-astronomy/staff/rse204">Ryan Edginton</a>, we used a portable spectrometer (Ocean Optics USB2000+VIS-NIR-ES, connected to a CC3 cosine corrector with a 3.9 mm collection diameter attached to a 0.55 mm diameter optical fibre) to measure light spectra and absolute intensity in the visible range.

Line 957:

<h6>Characterisation experiment</h6>

The following graphs show the average percentage viable cells for induced and uninduced samples after 6 hrs of exposure

−

to 1.2 µW/cm2 of white light and an average temperature of 38.63 °C. CFU count for the control condition was treated as 100 % and viable cells calculated

+

to 1.2 mW/cm2 of white light and an average temperature of 38.63 °C. CFU count for the control condition was treated as 100 % and viable cells calculated

as a proportion of that value. CFUs were not counted above 300. Error bars represent

the standard error of the mean. Little to no difference in CFU count is shown between the control (BL21 (DE3)) and kill switch samples when they are kept in the dark. There is a significant difference in the number of CFUs when the kill switch samples are exposed to light.

Line 1,021:

</div>

−

<h6>Ministat experiment</h6>

+

<h6 id="MSE">Ministat experiment</h6>

All samples from the ministat were tested using the KillerRed, KillerOrange protocol found <a href="#KRKOProt">

here</a>. Glycerol stocks were made from the samples taken at each time interval, testing was done using these glycerol stocks.

Line 1,057:

cell wall (Jollès and Jollès, 1984).

Lysozymes are commonly used in mass spectrometry for protein mass calibration and are also effective lysing agents

−

against ~~gram~~-positive and ~~gram~~-negative bacteria (Sigma aaldrich, 2016). UNICAMP-Brazil 2009 iGEM team used the Lysozyme Gallus gallus part BBa_K284001 previously and other lysis mechanisms have been used as kill switches by TU-Delft 2013, Newcastle 2010, Imperial College London 2011 and METU-Ankara 2011. As others team have used lysis mechanisms in their kill switches we thought Lysozyme (Gallus gallus)

+

against Gram-positive and to lesser extent, Gram-negative bacteria (Sigma aaldrich, 2016). UNICAMP-Brazil 2009 iGEM team used the Lysozyme Gallus gallus part BBa_K284001 previously and other lysis mechanisms have been used as kill switches by TU-Delft 2013, Newcastle 2010, Imperial College London 2011 and METU-Ankara 2011. As others team have used lysis mechanisms in their kill switches we thought Lysozyme (Gallus gallus)

would be a suitable candidate to test the effectiveness of lysis as a kill switch mechanism and investigate the

potential for HGT if lysis is successful. We added an OmpA signal peptide to Lysozyme C which

Line 1,119:

than is used in the literature (Sarkisyan et al, 2015). On investigation into the kind of light source that was needed to produce

the 1 W/cm2 of previous experiments (Bulina et al, 2005), it became clear that 1 W/cm2

−

was impractically bright and would generate large amounts of heat which would kill E. coli. We decided to use a much less powerful LED array that produces 1.2 µW/cm2 at the wavelengths most effective for KillerRed and KillerOrange (Fig. 7) and expose our samples to light for a greater length of time. We showed that this was still effective with an average

+

was impractically bright and would generate large amounts of heat which would kill E. coli. We decided to use a much less powerful LED array that produces 1.2 mW/cm2 at the wavelengths most effective for KillerRed and KillerOrange (Fig. 7) and expose our samples to light for a greater length of time. We showed that this was still effective with an average

survival rate in the + IPTG condition of 2.2% for KillerRed (Fig. 11) and 12.7 % for KillerOrange (Fig. 13). A wider range of exposure times

and light intensities would greatly improve the characterisation of these parts, unfortunately time limitations prevented

Line 1,129:

°C for 24 hrs before exposing the samples to light (Sarkisyan et al, 2015), the reason given for this was to "increase the fraction of mature protein". We tested the validity of this as cultures were incubated at 37 °C 220 rpm overnight not 4 °C and the phototoxicity of KillerRed and KillerOrange was still evident. The light box itself had a negative effect on E.

coli growth. In our experiment each sample was first diluted to 10-3,10-4 and 10-5 before exposure

−

to light. The dark condition for the control formed a lawn of bacteria on the agar plate regardless of the starting dilution factor, however in the light condition, the 10-3 dilution produced the same amount of colonies as the dark but in greater dilutions the number of colonies decreased. This decrease was not significant enough to have affected the results but it should be noted that exposure to 1.2 µW/cm2 for 6 hrs slows the growth rate of E. coli BL21 DE3.

+

to light. The dark condition for the control formed a lawn of bacteria on the agar plate regardless of the starting dilution factor, however in the light condition, the 10-3 dilution produced the same amount of colonies as the dark but in greater dilutions the number of colonies decreased. This decrease was not significant enough to have affected the results but it should be noted that exposure to 1.2 mW/cm2 for 6 hrs slows the growth rate of E. coli BL21 DE3.

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.

Line 1,146:

The samples of lysozyme were assayed in the same way after continuous culture and did show a decrease in lysozyme activity over time, however the original readings that were used as a comparison have an error of sufficient size that this is not conclusive.

The CFU count for lysozyme showed no difference from the control. Lysozyme added to a sample extra-cellularly was shown to

−

lyse all the cells in our HGT experiment, even though ~~gram~~-negative bacteria are partially protected from its action due to

+

lyse all the cells in our HGT experiment, even though Gram-negative bacteria are partially protected from its action due to

their outer membrane (Callewaert, 2008). Yet lysozyme produced intra-cellularly and targeted to the periplasm was not

effective. There may have been issues with translocation of the protein to the target area, however this seems unlikely

Difference between revisions of "Team:Exeter/Project"

Latest revision as of 02:30, 20 October 2016

Project Exepire

There’s no need for kill switches in the industry area that we operate. Even in the case where we might think there was a need for extra containment, the idea of putting in multiple systems to a production strain is concerning
-Dr Markus Gershater, Synthace Ltd

This is not a zero risk activity, on the other hand there are massive benefits that might be realised from GMOs. The only way I can see of moving us forward is to educate the public a bit more about the issues.
-Prof Richard Titball. University of Exeter

Continuous culture

Metabolic kill switch

Method

Metabolic Kill Switch: Results

Characterisation experiment

Ministat experiment

Enzymatic kill switch

Method

Enzymatic Kill switch: Results

Characterisation

Horizontal Gene Transfer

DNase

Discussion

Metabolic Kill Switch: KillerRed and KillerOrange.

Enzymatic Kill Switch: Lysozyme

Failsafe

Genome integration

DNA degradation

Ministat

References

Transformation of competent E. coli cells

KillerRed KillerOrange protocol

Ministat Protocol

HGT Protocol

Preparation of Competent Cells protocol

@@ Line 514: / Line 514: @@
      <div>
          <div class="collapse navbar-collapse" id="myNavbar">
-						<ul class="nav navbar-nav">
+									<ul class="nav navbar-nav">
 				<li><div id="links_drop" class="dropdown">
    <button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true">
@@ Line 523: / Line 523: @@
 <li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Project">Lab Project</a></li>
      <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Labbook">Lab Book</a></li>
+	<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Safety">Safety</a></li>
    </ul>
@@ Line 551: / Line 552: @@
 <li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Awards">Awards</a></li>
-<li><span style="margin:10px 0 30px 2px;padding:0;">Special pages</span></li>
+<li><span style="margin:10px 0 30px 2px;padding:0;"><u>Special pages</u></span></li>
 <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Silver">HP Silver</a></li>
 <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Gold">HP Gold</a></li>
@@ Line 563: / Line 564: @@
 			</ul>
-				<ul class="nav navbar-nav navbar-right">
+			<ul class="nav navbar-nav navbar-right" >
 					<li style="height:50px;width:48px;"class="soc_1">
 						<a href="https://www.youtube.com/channel/UC31qfG4hnm8gRHDCkrBtAiQ">
@@ Line 766: / Line 767: @@
   bubbled through flasks of water to hydrate it and then used to agitate the culture. Chambers were inoculated with
   freshly transformed <i>E. coli</i> 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
+  By simulating in miniature how a kill switch might behave in an industrial setting, the ministat provides a <a href="https://2016.igem.org/Team:Exeter/Proof">proof
-  of concept for how a kill switch might be maintained in larger chemostats during a continuous culture. A protocol
+  of concept</a> 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 <a href="#MinistatProt">here</a>
 </p>
@@ Line 833: / Line 834: @@
   which, when irradiated with green and blue light respectively, generate reactive oxygen species (ROS). KillerRed has been
   shown to effectively kill cells when exposed to green light (540–580 nm) and is much less effective under blue light
-  (460–490 nm) (Bulina <i>et al</i>, 2006). KillerOrange effectively kills cells when exposed to 450-495nm (Sarkisyan 2015), the range that KillerRed does not. The open Beta barrel of the protein is thought to allow the solvent to come into contact with the chromophore, facilitating the release of reactive oxygen species (Carpentier <i>et al</i>, 2009)</p>
+  (460–490 nm) (Bulina <i>et al</i>, 2006). KillerOrange effectively kills cells when exposed to 450-495nm (Sarkisyan 2015), the range that KillerRed does not. There is a β-barrel present in both these proteins. A water-filled channel that is in contact with a chromophore area and located at the cap of the said β-barrel is thought to confer these proteins with their phototoxic capabilities (Pletnev S <i>et al</i>, 2009).</p>
 <div class="row">
@@ Line 844: / Line 845: @@
 </div>
 <p id="pp">
-The mechanism by which ROS kill cells is not fully understood, partly because they react quickly
+The mechanism by which ROS kill cells isn’t completely understood. However ROS have been shown to have various detrimental roles in cells such as the oxidation of thiols, ascorbate and proteins containing (Fe-S)4 clusters as well as reducing various transition metals (Farr and Kogama, 1991). Prolonged exposure and or high levels of ROS triggers apoptosis like mechanisms (Held, 2015).<br>Our metabolic kill switches build on previous iGEM projects which have used the expression of highly phototoxic
- with contaminating metals to form more reactive species that obscure their own role in oxidation damage (Farr and Kogama, 1991). Prolonged exposure and or high levels of ROS triggers apoptosis like mechanisms (Held, 2015).<br>Our metabolic kill switches build on previous iGEM projects which have used the expression of highly phototoxic
 fluorescent proteins to kill the cells by exposing the culture to light. In 2013, the iGEM team from Carnegie Mellon developed a phage delivery system of the KillerRed gene, which was then expressed in the infected bacteria, killing it on exposure to light. Carnegie Mellon 2014 continued characterisation of KillerRed and its monomeric form Supernova by analysing their photobleaching characteristics. Neither team tested the longevity of the kill switch or provided details on the light intensity that the cultures were exposed to. We aim to quantify the length of time for which the kill switch remains viable and provide absolute values for the intensity of our light source.<br>
@@ Line 854: / Line 854: @@
 <h6>Method</h6>
-<p id="pp">The following samples were tested for phototoxicity by exposing them to 1.2 µW/cm<sup>2</sup> of white light from a 4x8 LED array (Fig. 6) for 6 hrs. Samples were then spread plated and colony forming units (CFUs) were counted. All parts were carried on the pSB1C3 plasmid and transformed into <i>E. coli</i> BL21 (DE3). Samples that were induced were done so with Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 nM.</p>
+<p id="pp">The following samples were tested for phototoxicity by exposing them to 1.2 mW/cm<sup>2</sup> of white light from a 4x8 LED array (Fig. 6) for 6 hrs. Samples were then spread plated and colony forming units (CFUs) were counted. All parts were carried on the pSB1C3 plasmid and transformed into <i>E. coli</i> BL21 (DE3). Samples that were induced were done so with Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 nM.</p>
 <p id="pp">Henceforth samples will be refered to as:</p>
 <style>
@@ Line 932: / Line 932: @@
 <br>
 </div>
-<p id="pp">KillerRed is excited by green/yellow light (540-580 nm) and KillerOrange by blue light (460-490 nm). We constructed a box around the LED array to prevent ambient light entering. Access to the inside of the box was gained through an opening cut in the front. With help from <a href="#">Ryan Edginton</a>, we used a portable spectrometer (Ocean Optics USB2000+VIS-NIR-ES, connected to a CC3 cosine corrector with a 3.9 mm collection diameter attached to a 0.55 mm diameter optical fibre) to measure light spectra and absolute intensity in the visible range. </p>
+<p id="pp">KillerRed is excited by green/yellow light (540-580 nm) and KillerOrange by blue light (460-490 nm). We constructed a box around the LED array to prevent ambient light entering. Access to the inside of the box was gained through an opening cut in the front. With help from <a href="http://emps.exeter.ac.uk/physics-astronomy/staff/rse204">Ryan Edginton</a>, we used a portable spectrometer (Ocean Optics USB2000+VIS-NIR-ES, connected to a CC3 cosine corrector with a 3.9 mm collection diameter attached to a 0.55 mm diameter optical fibre) to measure light spectra and absolute intensity in the visible range. </p>
 <div class="row">
      <div class="col-xs-6" style="padding:5px 2% 5px 10%;margin:0;">
@@ Line 957: / Line 957: @@
 <h6><u>Characterisation experiment</u></h6>
 <p id="pp">The following graphs show the average percentage viable cells for induced and uninduced samples after 6 hrs of exposure
-to 1.2 µW/cm<sup>2</sup> of white light and an average temperature of 38.63 °C. CFU count for the control condition was treated as 100 % and viable cells calculated
+to 1.2 mW/cm<sup>2</sup> of white light and an average temperature of 38.63 °C. CFU count for the control condition was treated as 100 % and viable cells calculated
 as a proportion of that value. CFUs were not counted above 300. Error bars represent
   the standard error of the mean. Little to no difference in CFU count is shown between the control (BL21 (DE3)) and kill switch samples when they are kept in the dark. There is a significant difference in the number of CFUs when the kill switch samples are exposed to light.
@@ Line 1,021: / Line 1,021: @@
      </div>
 </div>
-<h6><u>Ministat experiment</u></h6>
+<h6 id="MSE"><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 from the samples taken at each time interval, testing was done using these glycerol stocks.</p>
@@ Line 1,057: / Line 1,057: @@
   cell wall (Jollès and Jollès, 1984).
 Lysozymes are commonly used in mass spectrometry for protein mass calibration and are also effective lysing agents
-  against gram-positive and gram-negative bacteria (Sigma aaldrich, 2016). UNICAMP-Brazil 2009 iGEM team used the Lysozyme <i>Gallus gallus</i> part BBa_K284001 previously and other lysis mechanisms have been used as kill switches by TU-Delft 2013, Newcastle 2010, Imperial College London 2011 and METU-Ankara 2011. As others team have used lysis mechanisms in their kill switches we thought Lysozyme (<i>Gallus gallus</i>)
+  against Gram-positive and to lesser extent, Gram-negative bacteria (Sigma aaldrich, 2016). UNICAMP-Brazil 2009 iGEM team used the Lysozyme <i>Gallus gallus</i> part BBa_K284001 previously and other lysis mechanisms have been used as kill switches by TU-Delft 2013, Newcastle 2010, Imperial College London 2011 and METU-Ankara 2011. As others team have used lysis mechanisms in their kill switches we thought Lysozyme (<i>Gallus gallus</i>)
   would be a suitable candidate to test the effectiveness of lysis as a kill switch mechanism and investigate the
   potential for HGT if lysis is successful. We added an OmpA signal peptide to Lysozyme C which
@@ Line 1,119: / Line 1,119: @@
 than is used in the literature (Sarkisyan <i>et al</i>, 2015). On investigation into the kind of light source that was needed to produce
   the 1 W/cm<sup>2</sup> of previous experiments (Bulina <i>et al</i>, 2005), it became clear that 1 W/cm<sup>2</sup>
-  was impractically bright and would generate large amounts of heat which would kill <i>E. coli</i>. We decided to use a much less powerful LED array that produces 1.2 µW/cm<sup>2</sup> at the wavelengths most effective for KillerRed and KillerOrange (Fig. 7) and expose our samples to light for a greater length of time. We showed that this was still effective with an average
+  was impractically bright and would generate large amounts of heat which would kill <i>E. coli</i>. We decided to use a much less powerful LED array that produces 1.2 mW/cm<sup>2</sup> at the wavelengths most effective for KillerRed and KillerOrange (Fig. 7) and expose our samples to light for a greater length of time. We showed that this was still effective with an average
   survival rate in the + IPTG condition of 2.2% for KillerRed (Fig. 11) and 12.7 % for KillerOrange (Fig. 13). A wider range of exposure times
   and light intensities would greatly improve the characterisation of these parts, unfortunately time limitations prevented
@@ Line 1,129: / Line 1,129: @@
   °C for 24 hrs before exposing the samples to light (Sarkisyan <i>et al</i>, 2015), the reason given for this was to "increase the fraction of mature protein". We tested the validity of this as cultures were incubated at 37 °C 220 rpm overnight not 4 °C and the phototoxicity of KillerRed and KillerOrange was still evident. The light box itself had a negative effect on <i>E.
   coli</i> growth. In our experiment each sample was first diluted to 10<sup>-3</sup>,10<sup>-4</sup> and 10<sup>-5</sup> before exposure
-  to light. The dark condition for the control formed a lawn of bacteria on the agar plate regardless of the starting dilution factor, however in the light condition, the 10<sup>-3</sup> dilution produced the same amount of colonies as the dark but in greater dilutions the number of colonies decreased. This decrease was not significant enough to have affected the results but it should be noted that exposure to 1.2 µW/cm<sup>2</sup> for 6 hrs slows the growth rate of <i>E. coli</i> BL21 DE3.</p>
+  to light. The dark condition for the control formed a lawn of bacteria on the agar plate regardless of the starting dilution factor, however in the light condition, the 10<sup>-3</sup> dilution produced the same amount of colonies as the dark but in greater dilutions the number of colonies decreased. This decrease was not significant enough to have affected the results but it should be noted that exposure to 1.2 mW/cm<sup>2</sup> for 6 hrs slows the growth rate of <i>E. coli</i> BL21 DE3.</p>
 <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.
@@ Line 1,146: / Line 1,146: @@
   The samples of lysozyme were assayed in the same way after continuous culture and did show a decrease in lysozyme activity over time, however the original readings that were used as a comparison have an error of sufficient size that this is not conclusive.
   The CFU count for lysozyme showed no difference from the control. Lysozyme added to a sample extra-cellularly was shown to
-  lyse all the cells in our HGT experiment, even though gram-negative bacteria are partially protected from its action due to
+  lyse all the cells in our HGT experiment, even though Gram-negative bacteria are partially protected from its action due to
   their outer membrane (Callewaert, 2008). Yet lysozyme produced intra-cellularly and targeted to the periplasm was not
   effective. There may have been issues with translocation of the protein to the target area, however this seems unlikely

Difference between revisions of "Team:Exeter/Project"

Latest revision as of 02:30, 20 October 2016

Project Exepire

There’s no need for kill switches in the industry area that we operate. Even in the case where we might think there was a need for extra containment, the idea of putting in multiple systems to a production strain is concerning -Dr Markus Gershater, Synthace Ltd

This is not a zero risk activity, on the other hand there are massive benefits that might be realised from GMOs. The only way I can see of moving us forward is to educate the public a bit more about the issues. -Prof Richard Titball. University of Exeter

Continuous culture

Metabolic kill switch

Method

Metabolic Kill Switch: Results

Characterisation experiment

Ministat experiment

Enzymatic kill switch

Method

Enzymatic Kill switch: Results

Characterisation

Horizontal Gene Transfer

DNase

Discussion

Metabolic Kill Switch: KillerRed and KillerOrange.

Enzymatic Kill Switch: Lysozyme

Failsafe

Genome integration

DNA degradation

Ministat

References

Transformation of competent E. coli cells

KillerRed KillerOrange protocol

Ministat Protocol

HGT Protocol

Preparation of Competent Cells protocol

There’s no need for kill switches in the industry area that we operate. Even in the case where we might think there was a need for extra containment, the idea of putting in multiple systems to a production strain is concerning
-Dr Markus Gershater, Synthace Ltd

This is not a zero risk activity, on the other hand there are massive benefits that might be realised from GMOs. The only way I can see of moving us forward is to educate the public a bit more about the issues.
-Prof Richard Titball. University of Exeter