Difference between revisions of "Team:Exeter/Project"
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<img src="https://static.igem.org/mediawiki/2016/d/d0/T--Exeter--KOcont.jpg" | <img src="https://static.igem.org/mediawiki/2016/d/d0/T--Exeter--KOcont.jpg" | ||
style="max-width:100%;margin:auto;display:block;"> | style="max-width:100%;margin:auto;display:block;"> | ||
| − | <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 | + | <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> | ||
<br> | <br> | ||
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<p id="pp">Lysozymes are a group of enzymes that are an important part of the immune response against bacteria | <p id="pp">Lysozymes are a group of enzymes that are an important part of the immune response against bacteria | ||
| − | (Myrnes et al, 2013). They are defined as 1,4-fl-N-acetylmuramidases that cleave the glycosidic | + | (Myrnes et al, 2013). They are defined as 1,4-fl-N-acetylmuramidases that cleave the glycosidic bonds between carbon 1 of N-acetylmuramic acid and carbon 4 of N-acetylglucosamine in the peptidoglycan that makes up a bacterial |
| − | carbon 1 of N-acetylmuramic acid and | + | cell wall (Jollès and Jollès, 1984). |
| − | cell wall (Jollès and Jollès, 1984) | + | |
Lysozymes are commonly used in mass spectrometry for protein mass calibration and are also effective lysing agents | 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 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 we thought Lysozyme (<i>Gallus gallus</i>) | + | 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>) |
would be a suitable candidate to test the effectiveness of lysis as a kill switch mechanism and investigate the | 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 | potential for HGT if lysis is successful. We added an OmpA signal peptide to Lysozyme C which | ||
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to lysozyme activity in the sample. The fluorescence assay was used to measure the activity of the freshly | to lysozyme activity in the sample. The fluorescence assay was used to measure the activity of the freshly | ||
transformed kill switch and that of the cultures grown in the ministat. CFUs were also used as a measure of | transformed kill switch and that of the cultures grown in the ministat. CFUs were also used as a measure of | ||
| − | efficiency by comparing number of colonies to a control. 5 ml ovenights of <i>E. coli</i> BL21 (DE3) transformed | + | efficiency by comparing the number of colonies to a control. 5 ml ovenights of <i>E. coli</i> BL21 (DE3) transformed |
with pSB1C3 lysozyme were used to inoculate 250 ml Erlenmeyer flasks containing 50 ml of LB laced with 35 µg/ml | with pSB1C3 lysozyme were used to inoculate 250 ml Erlenmeyer flasks containing 50 ml of LB laced with 35 µg/ml | ||
chloramphenicol. Once an OD of 0.23 was reached IPTG was added to a final concentration of 0.2 nM. Protein production | chloramphenicol. Once an OD of 0.23 was reached IPTG was added to a final concentration of 0.2 nM. Protein production | ||
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<h3>DNase</h3> | <h3>DNase</h3> | ||
| − | <p id="pp"> | + | <p id="pp">DNase I is a nonspecific deoxyribonuclease originally extracted from bovine pancreatic tissue. It degrades both |
double-stranded and single-stranded DNA resulting in the release of di-, tri- and oligonucleotide products with 5´ | double-stranded and single-stranded DNA resulting in the release of di-, tri- and oligonucleotide products with 5´ | ||
| − | -phosphorylated and 3´-hydroxylated ends (Vanecko, 1961). | + | -phosphorylated and 3´-hydroxylated ends (Vanecko, 1961). DNase I has also been shown to work on chromatin and DNA:RNA |
| − | hybrids (Kunitz, 1950). | + | hybrids (Kunitz, 1950). DNase I degrades these target polymer molecules through the hydrolytic cleavage of phosphodiester linkages in their backbone (Suck, 1986).</p> |
| − | + | ||
| − | + | ||
<p id="pp">For a kill switch to be effective as a bio-containment device, the release of synthetic DNA must be mitigated. | <p id="pp">For a kill switch to be effective as a bio-containment device, the release of synthetic DNA must be mitigated. | ||
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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 | 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 | and light intensities would greatly improve the characterisation of these parts, unfortunately time limitations prevented | ||
| − | us from testing this.<br><br> The leakiness of the T7 promoter used in our kill switches was quantified by comparing protein production in the + IPTG condition and - IPTG condition. A one tail t-test assuming equal variance was performed for the mean fluorescence values of the cultures tested in the light box. Fluorescence was used as a measure of protein production. No statistically significant difference was found between the + IPTG condition and – IPTG condition (a significance value of < 0.05 | + | us from testing this.<br><br> The leakiness of the T7 promoter used in our kill switches was quantified by comparing protein production in the + IPTG condition and - IPTG condition. A one tail t-test assuming equal variance was performed for the mean fluorescence values of the cultures tested in the light box. Fluorescence was used as a measure of protein production. No statistically significant difference was found between the + IPTG condition and – IPTG condition (a significance value of < 0.05 was used. p-value for KillerRed 0.18, p-value for KillerOrange 0.16). CFU counts |
for + IPTG conditions were within the standard error of – IPTG (Fig. 11 & 13). For KillerRed the induced kill switch appears to be more | for + IPTG conditions were within the standard error of – IPTG (Fig. 11 & 13). For KillerRed the induced kill switch appears to be more | ||
effective whereas the uninduced switch is more effective in KillerOrange. The leakiness of the T7 promoter has likely | effective whereas the uninduced switch is more effective in KillerOrange. The leakiness of the T7 promoter has likely | ||
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°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. | °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 | 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 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 | + | 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> |
<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. | <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. | ||
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being produced, up regulation of native <i>E. coli</i> enzymes that mitigate the effects of ROS may be the cause of the increase | 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 | 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.</p> | + | 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> |
<h6>Enzymatic Kill Switch: Lysozyme</h6> | <h6>Enzymatic Kill Switch: Lysozyme</h6> | ||
<p id="pp">The Enzcheck fluorescence assay used to determine lysozyme activity produced values that were not consistent | <p id="pp">The Enzcheck fluorescence assay used to determine lysozyme activity produced values that were not consistent | ||
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The assay showed that 20x diluted sample produced near 500 U/ml activity readings yet this culture would still produce | The assay showed that 20x diluted sample produced near 500 U/ml activity readings yet this culture would still produce | ||
a lawn of bacteria when 200 µl was spread plated. It is noted that the standard curve was of poor quality. | a lawn of bacteria when 200 µl was spread plated. It is noted that the standard curve was of poor quality. | ||
| − | The samples of lysozyme were assayed in the same way after continuous culture and did show a decrease in lysozyme activity, | + | 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 | 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 | + | 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 | effective. There may have been issues with translocation of the protein to the target area, however this seems unlikely | ||
due to the effectiveness of its extracellular action. Another explanation may be that lysozyme as a kill switch mechanism | due to the effectiveness of its extracellular action. Another explanation may be that lysozyme as a kill switch mechanism | ||
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<h3>Failsafe</h3> | <h3>Failsafe</h3> | ||
| − | <p id="pp"> One area that we were unable to explore was the | + | <p id="pp"> One area that we were unable to explore was the incorporation of multiple kill switches into the same system. |
Initially we aimed to construct an operon that contained KillerRed and KillerOrange. This was unfeasible with the cloning | Initially we aimed to construct an operon that contained KillerRed and KillerOrange. This was unfeasible with the cloning | ||
strategy that we were using as the overhangs that join the ribosome binding site (RBS) to the CDS would not differentiate between KillerRed and | strategy that we were using as the overhangs that join the ribosome binding site (RBS) to the CDS would not differentiate between KillerRed and | ||
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<p id="pp">While plasmids are widely used to carry genetic parts, integration into the host genome | <p id="pp">While plasmids are widely used to carry genetic parts, integration into the host genome | ||
| − | could prove a more robust approach to introducing genes into | + | could prove a more robust approach to introducing genes into an organism. Genome |
integration removes the need for a selectable antibiotic resistance marker as parts | integration removes the need for a selectable antibiotic resistance marker as parts | ||
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investigate whether integration into the <i>E. coli</i> genome will affect the efficiency of our | investigate whether integration into the <i>E. coli</i> genome will affect the efficiency of our | ||
| − | kill switches and whether they | + | kill switches and whether they would remain functional for longer in a continuous culture. We aimed to use the lambda red |
recombination method to integrate our parts into the <i>ars</i>B locus using the pKD4 plasmid as a vector. Integrating at | recombination method to integrate our parts into the <i>ars</i>B locus using the pKD4 plasmid as a vector. Integrating at | ||
<i>ars</i>B has been shown not to affect <i>E. coli</i> growth (Sabri <i>et al</i>, 2013). However the pKD4 plasmid contained | <i>ars</i>B has been shown not to affect <i>E. coli</i> growth (Sabri <i>et al</i>, 2013). However the pKD4 plasmid contained | ||
illegal EcoRI and XBal restriction sites. | illegal EcoRI and XBal restriction sites. | ||
| − | To resolve this we decided to carry out site directed mutagenesis to change one nucleotide | + | To resolve this we decided to carry out site directed mutagenesis to change one nucleotide in each sequence |
of the restriction sites. Primers were designed for use with the Q5 site directed mutagenesis kit. The first attempt | of the restriction sites. Primers were designed for use with the Q5 site directed mutagenesis kit. The first attempt | ||
using this kit involved a 2 step PCR reaction, this was shown by gel electrophoresis of the product to have been | using this kit involved a 2 step PCR reaction, this was shown by gel electrophoresis of the product to have been | ||
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<p id="pp">We had hoped to develop a CRISPR based kill switch building on the work of Caliando and Voigt (2015). | <p id="pp">We had hoped to develop a CRISPR based kill switch building on the work of Caliando and Voigt (2015). | ||
We designed the spacer array to target three essential genes <i>polA</i>, <i>rpoC</i> and <i>topA</i> using the | We designed the spacer array to target three essential genes <i>polA</i>, <i>rpoC</i> and <i>topA</i> using the | ||
| − | deskgen platform. We selected three protospacers within the CDS of each essential gene. The cleavage sites were | + | <i>deskgen</i> platform. We selected three protospacers within the CDS of each essential gene. The cleavage sites were |
designed to be in the first third, the centre third and the final third of the CDS. The spacer array was designed | designed to be in the first third, the centre third and the final third of the CDS. The spacer array was designed | ||
| − | to be carried on the pSB1C3 plasmid under the control of a constituitive promoter (BBa_J23100). Our aim was | + | to be carried on the pSB1C3 plasmid under the control of a constituitive promoter (BBa_J23100). Our aim was to |
investigate how many essential genes would be needed for the kill switch to be effective, whether some genes | investigate how many essential genes would be needed for the kill switch to be effective, whether some genes | ||
were more effective targets than others and whether targeting multiple protospacers simultaneously was more | were more effective targets than others and whether targeting multiple protospacers simultaneously was more | ||
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by Caliando and Voigt (2015) to be stable for many months when integrated into the genome at multiple loci. Making this | by Caliando and Voigt (2015) to be stable for many months when integrated into the genome at multiple loci. Making this | ||
system available to iGEM teams could greatly improve on the short comings we have shown in the stability of toxic protein | system available to iGEM teams could greatly improve on the short comings we have shown in the stability of toxic protein | ||
| − | based switches carried on plasmids.</p> | + | based switches carried on plasmids. If a system of this kind were to target just the synthetic DNA that had been introduced into the organism, the system would prevent release of synthetic DNA into the environment without the high selection pressure of death. This would potentially allow the switch to be retained for longer. </p> |
<h3>Ministat</h3> | <h3>Ministat</h3> | ||
<p id="pp">The modularity of the ministat allows several environmental conditions to be tested simultaneously. | <p id="pp">The modularity of the ministat allows several environmental conditions to be tested simultaneously. | ||
Revision as of 22:24, 18 October 2016
