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Team:Arizona State/ssDNA
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| - | + | The wild type form of topoisomerase binds to the DNA sequence (YCCTT) in E. Coli. It regulates the winding of the DNA by making a nick after the second T. This allows for the rotation of the strands to relieve torsional stress. Afterwards, the DNA strands are religated again. In 2006, Bushman et al. have shown that the smallpox topoisomerase double cysteine mutant D168A mutates the tyrosine responsible for covalent bonding to the 5’ phosphate at the DNA nicking. This mutant form prevents religation, and thus causes the majority of the DNA to stay in the covalently bonded complex. | |
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| + | In our design, we plan to use topoisomerase to nick a specific covalently bonded sequence and peel off a section of single stranded DNA. We have designed a template plasmid that includes tandem YCCTT recognition sites with template strand in between, and is complementary to a section of coding sequence of GFP. We plan to use a KEIO strain with one copy of this coding sequence in the E. Coli genome. | ||
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| + | Basilion et al. from Case Western in 2010 have shown that they were able to make a split beta-gal complementation assay with relatively reliable assay results In the assay, alpha-4 / omega, which has a higher specificity, is the most successful split beta assay. It is thus used to eliminate false positive. Additionally, we are adapting alpha and 1-omega, which is less specific but has a higher signal, for the same protocol to eliminate false negative. | ||
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| + | Additionally, Basilion et al. also demonstrated success in creating fusion proteins with a split-beta gal fragment and antibody specific to their target. Modifying this, we plan to make a fusion of our mutant topoisomerase and our split-beta gal fragments. This effectively creates a probe that when assembled contains topoisomerase bound both to a single stranded DNA hybridization probe and a split-beta gal fragment. By incubating the two probes that recognize adjacent DNA sequences, we can test for the presence of DNA sequences in a bacterial genome. | ||
Revision as of 08:27, 3 October 2012
The wild type form of topoisomerase binds to the DNA sequence (YCCTT) in E. Coli. It regulates the winding of the DNA by making a nick after the second T. This allows for the rotation of the strands to relieve torsional stress. Afterwards, the DNA strands are religated again. In 2006, Bushman et al. have shown that the smallpox topoisomerase double cysteine mutant D168A mutates the tyrosine responsible for covalent bonding to the 5’ phosphate at the DNA nicking. This mutant form prevents religation, and thus causes the majority of the DNA to stay in the covalently bonded complex.
In our design, we plan to use topoisomerase to nick a specific covalently bonded sequence and peel off a section of single stranded DNA. We have designed a template plasmid that includes tandem YCCTT recognition sites with template strand in between, and is complementary to a section of coding sequence of GFP. We plan to use a KEIO strain with one copy of this coding sequence in the E. Coli genome.
Basilion et al. from Case Western in 2010 have shown that they were able to make a split beta-gal complementation assay with relatively reliable assay results In the assay, alpha-4 / omega, which has a higher specificity, is the most successful split beta assay. It is thus used to eliminate false positive. Additionally, we are adapting alpha and 1-omega, which is less specific but has a higher signal, for the same protocol to eliminate false negative.
Additionally, Basilion et al. also demonstrated success in creating fusion proteins with a split-beta gal fragment and antibody specific to their target. Modifying this, we plan to make a fusion of our mutant topoisomerase and our split-beta gal fragments. This effectively creates a probe that when assembled contains topoisomerase bound both to a single stranded DNA hybridization probe and a split-beta gal fragment. By incubating the two probes that recognize adjacent DNA sequences, we can test for the presence of DNA sequences in a bacterial genome.
