Revision as of 09:41, 3 October 2012

Hello! iGEM Calgary's wiki functions best with Javascript enabled, especially for mobile devices. We recommend that you enable Javascript on your device for the best wiki-viewing experience. Thanks!

Kill Genes: An active approach

Background

In our active kill switch, two novel nuclease enzymes work in tandem to cause fine degradation of the bacterial genome.

Nuclease genes in the 2011 parts registry were insufficient for design parameters in our project for two reasons: one, these enzymes did not provide sufficiently fine genome degradation; and two, the operating temperatures of these enzymes are unsuitable for tailings pond conditions. To this end, we submitted S7 micrococcal nuclease and CviAII into the parts registry.

S7 micrococcal nuclease

S7 nuclease is native to Staphylococcus aureus. S. aureus 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.

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). Henceforth, the CviAII restriction site is 16 times more prevalent in the E. coli genome and causes finer 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 CviAII functionality is 23 degrees Celsius (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 operating temperatures.

Nuclease assay: our nucleases versus those in the 2011 registry (BglII and BamHI):

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 E. coli 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 E. coli 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.

Figure X:

@@ Line 7: / Line 7: @@
 <h2>Background</h2>
-<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>
+<p>In our active kill switch, two novel nuclease enzymes work in tandem to cause fine degradation of the bacterial genome.</p>
-<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>
+<p>Nuclease genes in the 2011 parts registry were insufficient for design parameters in our project for two reasons: one, these enzymes did not provide sufficiently fine genome degradation; and two, the operating temperatures of these enzymes are unsuitable for tailings pond conditions. To this end, we submitted S7 micrococcal nuclease and CviAII into the parts registry.</p>
 <p></p>
@@ Line 17: / Line 17: @@
 <h2>CviAII restriction enzyme</h2>
-<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>
+<p>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). Henceforth, the CviAII restriction site is 16 times more prevalent in the E. coli genome and causes finer 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 CviAII functionality is 23 degrees Celsius (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 operating temperatures.</p>
-<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>
+<h3>Nuclease assay: our nucleases versus those in the 2011 registry (BglII and BamHI):</h3>
-<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>
-<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>
-<h3>Nuclease assay to evaluate the nucleases present in the registry (BglII and BamHI):</h3>
 <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.
 </html>[[File:UCalgary2012 RE-S7&amp;CviaII.png|thumb|300px|left|Figure X: ]]<html>

Team:Calgary/Project/HumanPractices/Killswitch/KillGenes

From 2012.igem.org