"
Team:Calgary/Project/HumanPractices/Killswitch/KillGenes
From 2012.igem.org
| Line 12: | Line 12: | ||
</html>[File:Bamandbgl uclagary.png|centre|thumb|350px|Figure 1. Comparison of the number of cut sites of various enzymes within the MG1655 genome.]]<html> | </html>[File:Bamandbgl uclagary.png|centre|thumb|350px|Figure 1. Comparison of the number of cut sites of various enzymes within the MG1655 genome.]]<html> | ||
| - | |||
<h2>Kill Gene: S7 micrococcal nuclease</h2> | <h2>Kill Gene: S7 micrococcal nuclease</h2> | ||
<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. </p> | <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. </p> | ||
Revision as of 20:14, 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
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 and CviAII. 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). [File:Bamandbgl uclagary.png
