Team:Calgary/Project/HumanPractices/Killswitch/KillGenes

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<h2>Background</h2>
<h2>Background</h2>
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<p>The principal mechanism behind our active killswitch system are exo and endonucleases which work in tandem to cause substantial degradation of the bacterial genome. The chance event of bacterial escape from the bioreactor into tailings ponds triggers the transcription of S7 micrococcal nuclease and CviAII endonuclease.</p>
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<p>When designing our actual kill genes, we needed to consider again the challenges of our environment. Many of the restriction enzymes found in the registry such as BamHI and BglII are active only at temperatures around 37&deg; C.  Although our bioreactor may be at this temperature, the surrounding environment would be much cooler. Since the kill genes <i>should</i> be active in the surrounding environment, we needed to pick enzymes that would be active at lower temperatures. In addition to that we also wanted enzymes that would cut very frequently in the <i>E. coli</i> genome to limit the chance of horizontal gene transfer.  Finally, as we chose to use an inducible system, which can easily be mutated, we wanted to introduce some redundancy by using two different kill genes. </p>
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<p>At the outset of this project, we considered using nucleases which were already present in the registry. Nuclease genes currently in the parts registry proved insufficient for our design parameters for two primary reasons: one, the recognition sites of these enzymes were not frequent enough for widespread genome degradation; and two, the operating temperatures of these enzymes were not optimal for tailings pond conditions. Henceforth, we submitted two new nuclease genes into the registry.</p>
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<p> We ended up choosing genes for two novel kill enzymes: S7 micrococcal nuclease (<a href="http://partsregistry.org/Part:BBa_K902019">BBa_K902019</a>) and CviAII (<a href="http://partsregistry.org/Part:BBa_K902022">BBa_K902022</a>).  Both of these enzymes are active at much lower temperatures than restriction enzymes. Through sequence analysis of the <i>E. coli</i> genome, we also determined that they cut extremely frequently in the genome, much more so than BglII or BamHI even combined (<b>Figure 1</b>).
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</html>[[File:Bamandbgl uclagary.png|centre|thumb|550px|Figure 1: Comparison of the number of cut-sites for BamHI, BglII, and CviAII nucleases within the ''E. coli'' strain K12 genome.]]<html>
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<h2>S7 micrococcal nuclease</h2>
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<p>S7 nuclease is native to <i>Staphylococcus aureus. S. aureus </i> uses this enzyme to destroy extracellular DNA when it infects humans. S7 has both endo and exonuclease activity. This enzyme has a preference for -AT rich regions as opposed to -GC rich regions. However, this enzyme digests the DNA into <200 bp fragments. Ideally this enzyme will be present both intracellularly and extracellularly.  We synthesized this enzyme from IDT. However this came with a mutation which altered a lysine residue to an isoleucine thereby making the enzyme dysfunctional. </p>
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<h2>Kill Gene: S7 micrococcal nuclease</h2>
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<p>S7 nuclease is native to <i>Staphylococcus aureus. S. aureus </i> uses this enzyme to destroy extracellular DNA when it infects humans to evade the immune system. S7 has both endo and exonuclease activity, and has a preference for -AT rich regions as opposed to -GC rich regions. Due to the non-specificity and high activity of this enzyme, it digests the DNA into <200 bp fragments. Ideally this enzyme will be present both intracellularly and extracellularly.  The intracellular fraction would degrade the <i>E. coli</i> genome and the extracellular fraction would degrade any free floating DNA  thereby reducing the chances of horizontal gene transfer. We synthesized this enzyme from IDT, however it came with a mutation which altered a lysine residue to an isoleucine making the enzyme dysfunctional. In order to overcome this issue, constructs with S7 were subjected to site-directed mutagenesis to restore the activity of the enzyme (Dingwall <i>et al</i>., 1981). </p>
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<h2>CviAII restriction enzyme</h2>
 
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<p>CviAII is a restriction endonuclease that was sourced from the Chlorella virus PBCV-1 (REF PAPER). Our team selected this enzyme for three reasons.</p>
 
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<p>Firstly, this enzyme recognizes the small four-base restriction site CATG wherein it cuts a staggered end between the A and C on the forward and reverse strands. This is advantageous for the design of our system because of the frequency of this short cut site in the E. coli genome. As opposed to the six base cutter BamHI system submitted by the 2007 Berkely team (BBa_I716462), the CviAII restriction site is to be 16 times more prevalent in the E. coli genome, which translates in finder degradation of the genetic material.</p>
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<h2>Kill Gene: CviAII Restriction Enzyme</h2>
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<p>CviAII is a restriction endonuclease that was sourced from the <i>Chlorella</i> virus PBCV-1 (Zhang <i>et al</i>., 1992). Our team selected this enzyme for three reasons. Firstly, this enzyme recognizes small, four-base pair restriction sites as opposed to other restriction enzymes such as the six-base cutter BamHI from the 2007 Berkely team (<a href="http://partsregistry.org/Part:BBa_I716462">BBa_I716462</a>). Because of this, the CviAII restriction site is 16 times more prevalent in the E. coli genome and causes more thorough degradation of genetic material. Secondly, CviAII is able to cut Dam and Dcm methylated sites in the <i>E. coli</i> genome, and this decreased selectivity increases prevalence of cut sites. Finally, the temperature optimum for the enzyme is 23&deg;C (Zhang <i>et al</i>., 1992). This optimum is closer to temperature conditions in the tailings ponds, and thus, CviAII will exhibit better enzyme activity as opposed to other enzymes in the registry with higher optimal temperatures.</p>
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<p>Secondly, the CATG cut site has the probability of being present in start codons of one quarter of genes in the E. coli genome. As such, coding genes will preferentially be selected with activation of CviAII; at this point, the exonuclease activity of S7 micrococcal nuclease can complete degradation of the gene element. Additionally, CviAII is able to cut Dam and Dcm methylated sites in the E. coli genome, and this translates into decreased selectivity of the enzyme.</p>
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<h2>Nuclease Assay</h2>
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</html>[[File:UCalgary2012 RE-S7&amp;CviaII.png|thumb|300px|right|Figure 2: This assay compares the enzymes present in the regitry i.e, BglII and BamHI to the enzymes added by us, S7 and CviAII. This shows that S7 and CviAII degrade the DNA much quicker than BglII and BamHI combined.]]<html>  
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<p>Finally, the temperature optimum of CviAII functionality is 23 degrees Celsius (REF PAPER). This value is relatively low and better suited to operation the cooler tailings water compared to other systems in the registry. For example, the 2007 Berkely BamHI system is optimized for 37 degrees Celsius and thus would be non-functional the tailings ponds. FIND SOME DATA OF TAILING WATER TEMP. </p>
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<p>In order to compare the S7 and CviAII to other nucleases in the 2011 registry (BglII and BamHI), we used combinations of commercial enzymes from New England Biolabs to digest <i>E. coli</i> genomic preparations. Please view the <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/nucleaseassay">nuclease assay protocol</a> for more details on how this was done. Activity of S7 nuclease is extremely rapid and shows degradation at the zero time point (<b>Figure 2</b>). Following 45 minutes of incubation time S7 and CviAII have digested the <i>E. coli</i> genome into small fragments, whereas BamHI and BglII treated fragments are significantly larger. After 90 minutes, S7 and CviAII have sheared the genome into very small fragments (less than 200 bp in size) while there are no difference in the lanes with BglII and BamHI which are similar to the 45 minutes time point (<b>Figure 2</b>).
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<h3>Nuclease assay to evaluate the nucleases present in the registry (BglII and BamHI):</h3>
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<P> To compare S7 and CviAII to the nucleases already present in the registry we did a nuclease assay with commercially available enzymes from New England Biolabs and an <i>E. coli</i> genomic prep. To see detailed protocol please link see here/link. As can be seen in Figure X, S7 starts acting almost immediately. Within 45 minutes both S7 and CviAII have chewed up the <i>E. coli</i> genome into small fragments whereas BamHI and BglII have sheared the genome into large fragments. Additionally, in 90 minutes, S7 and CviAII have sheared the genome into pieces <200 bp in size whereas there is no difference in the lanes with BglII and BamHI at 90 minutes compared to 45 minutes.
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</html>[[File:UCalgary2012 RE-S7&amp;CviaII.png|thumb|300px|left|Figure X: ]]<html>
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Latest revision as of 05:49, 24 October 2012

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Kill Genes: An active approach

Background

When designing our actual kill genes, we needed to consider again the challenges of our environment. Many of the restriction enzymes found in the registry such as BamHI and BglII are active only at temperatures around 37° C. Although our bioreactor may be at this temperature, the surrounding environment would be much cooler. Since the kill genes should be active in the surrounding environment, we needed to pick enzymes that would be active at lower temperatures. In addition to that we also wanted enzymes that would cut very frequently in the E. coli genome to limit the chance of horizontal gene transfer. Finally, as we chose to use an inducible system, which can easily be mutated, we wanted to introduce some redundancy by using two different kill genes.

We ended up choosing genes for two novel kill enzymes: S7 micrococcal nuclease (BBa_K902019) and CviAII (BBa_K902022). Both of these enzymes are active at much lower temperatures than restriction enzymes. Through sequence analysis of the E. coli genome, we also determined that they cut extremely frequently in the genome, much more so than BglII or BamHI even combined (Figure 1).

Figure 1: Comparison of the number of cut-sites for BamHI, BglII, and CviAII nucleases within the E. coli strain K12 genome.

Kill Gene: S7 micrococcal nuclease

S7 nuclease is native to Staphylococcus aureus. S. aureus uses this enzyme to destroy extracellular DNA when it infects humans to evade the immune system. S7 has both endo and exonuclease activity, and has a preference for -AT rich regions as opposed to -GC rich regions. Due to the non-specificity and high activity of this enzyme, it digests the DNA into <200 bp fragments. Ideally this enzyme will be present both intracellularly and extracellularly. The intracellular fraction would degrade the E. coli genome and the extracellular fraction would degrade any free floating DNA thereby reducing the chances of horizontal gene transfer. We synthesized this enzyme from IDT, however it came with a mutation which altered a lysine residue to an isoleucine making the enzyme dysfunctional. In order to overcome this issue, constructs with S7 were subjected to site-directed mutagenesis to restore the activity of the enzyme (Dingwall et al., 1981).

Kill Gene: CviAII Restriction Enzyme

CviAII is a restriction endonuclease that was sourced from the Chlorella virus PBCV-1 (Zhang et al., 1992). Our team selected this enzyme for three reasons. Firstly, this enzyme recognizes small, four-base pair restriction sites as opposed to other restriction enzymes such as the six-base cutter BamHI from the 2007 Berkely team (BBa_I716462). Because of this, the CviAII restriction site is 16 times more prevalent in the E. coli genome and causes more thorough degradation of genetic material. Secondly, CviAII is able to cut Dam and Dcm methylated sites in the E. coli genome, and this decreased selectivity increases prevalence of cut sites. Finally, the temperature optimum for the enzyme is 23°C (Zhang et al., 1992). This optimum is closer to temperature conditions in the tailings ponds, and thus, CviAII will exhibit better enzyme activity as opposed to other enzymes in the registry with higher optimal temperatures.

Nuclease Assay

Figure 2: This assay compares the enzymes present in the regitry i.e, BglII and BamHI to the enzymes added by us, S7 and CviAII. This shows that S7 and CviAII degrade the DNA much quicker than BglII and BamHI combined.

In order to compare the S7 and CviAII to other nucleases in the 2011 registry (BglII and BamHI), we used combinations of commercial enzymes from New England Biolabs to digest E. coli genomic preparations. Please view the nuclease assay protocol for more details on how this was done. Activity of S7 nuclease is extremely rapid and shows degradation at the zero time point (Figure 2). Following 45 minutes of incubation time S7 and CviAII have digested the E. coli genome into small fragments, whereas BamHI and BglII treated fragments are significantly larger. After 90 minutes, S7 and CviAII have sheared the genome into very small fragments (less than 200 bp in size) while there are no difference in the lanes with BglII and BamHI which are similar to the 45 minutes time point (Figure 2).