Difference between revisions of "Team:Exeter/Project"

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<h3>DNase</h3>
 
<h3>DNase</h3>
<p id="pp">For a kill switch to be effective as a bio-containment device, the release of synthetic DNA must be mitigate. We aimed to do this is our project using the expression of DNase I. Dnase I is commonly used in a laboratory setting to degrade unwanted DNA. It was shown by Worrall and Connolly (1990) that expression of DNase I is possible in <i>E. coli</i> as long as it is under the control of a promoter with a strong off state. We constructed a part with DNase I under the control of the T7 promoter. Unfortunately no transformations were successful and all colonies produced contained empty plasmid backbone. Worral and Connolly reported that a promoter which is less tightly regulated (pKK223-3) would result in transformation failure. As was shown in our metabolic kill switch, the T7 promoter we used to control expression of the CDS is very leaky. This is likely the reason why transformations were unsuccesful. As immediately after transformation, production of DNase I would commence killing all the cells. If this is the case future work on a system that uses DNase I as a kill switch but under much tighter control may prove very effective. <p>
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<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´-phosphorylated and 3´-hydroxylated ends (Vanecko, 1961). DNAse I has also been shown to work on chromatin and DNA:RNA hybrids (Kunitz, 1950).
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DNAse I degrades these target polymer molecules through the hydrolytic cleavage of phosphodiester linkages in their backbone (Suck, 1986).
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For a kill switch to be effective as a bio-containment device, the release of synthetic DNA must be mitigate. We aimed to do this is our project using the expression of DNase I. Dnase I is commonly used in a laboratory setting to degrade unwanted DNA. It was shown by Worrall and Connolly (1990) that expression of DNase I is possible in <i>E. coli</i> as long as it is under the control of a promoter with a strong off state. We constructed a part with DNase I under the control of the T7 promoter. Unfortunately no transformations were successful and all colonies produced contained empty plasmid backbone. Worral and Connolly reported that a promoter which is less tightly regulated (pKK223-3) would result in transformation failure. As was shown in our metabolic kill switch, the T7 promoter we used to control expression of the CDS is very leaky. This is likely the reason why transformations were unsuccesful. As immediately after transformation, production of DNase I would commence killing all the cells. If this is the case future work on a system that uses DNase I as a kill switch but under much tighter control may prove very effective. <p>
  
 
<h3>Discussion</h3>
 
<h3>Discussion</h3>

Revision as of 11:48, 12 October 2016