Team:Arizona State/Data

From 2012.igem.org

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Note from Dr. Haynes: Hi Nisarg<br>
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<html>
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<body>
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<h1>Data</h1>
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<hr style="color: #800000; height:3px;" />
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Please add an outline of the experiments-in-progress on the "Data" page. For instance…
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<h2>Topoisomerase-based DNA Biosensor</h2>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2012/8/8f/TopoDiagram.png" width="800" height="500" />
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</div>
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Experiment 1<br>
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<h3>Data For Our New Favorite Parts</h3>
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Aim: To determine that the split Beta-gal part of the biosensor works<br>
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<p>
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Approach: Co-express different versions of the alpha and omega fragments in E. coli<br>
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<a href="http://partsregistry.org/Part:BBa_K891234">D168A Double Cysteine Mutant of Smallpox Topoisomerase, BBa_K891234</a>
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Expected Results: If it works as expected, alpha and omega pairs will give a blue phenotype, and single units (negative controls) will not<br>
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</p>
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Lead experimenter: Abhi<br>
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<p>
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Progress: (to be filled in by Abhi)
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This mutant version of topoisomerase recognizes the YCCTT motif in dsDNA. It cleaves after the last T in this motif, making a single stranded nick, and covalently binds to the 3' phosphate on that thymine.
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</p>
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<p>
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<a href="http://partsregistry.org/Part:BBa_K891000">GFPT1, BBa_K891000</a>
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</p>
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<p>
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This part should be paired with GFPT2. This part codes for a 20bp sequence that is complementary to a portion of the genomic GFP coding sequence in <i>E.coli</i> Keio strains.
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</p>
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<p>
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<a href="http://partsregistry.org/Part:BBa_K891999">GFPT2, BBa_K891999</a>
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</p>
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<p>
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This part should be paired with GFPT1. This part codes for a 20bp sequence that is complementary to a portion of the genomic GFP coding sequence that comes after the GFPT1 binding site in <i>E.coli</i> Keio strains.
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</p>
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<br />
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<h2>Split Beta-Galactosidase Reporter System</h2>
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Experiment 2<br>
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<p>
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Aim: To quantitate Topo enzyme DNA nicking using a crazy sequencing approach<br>
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Tested alpha fragment of beta-galactosidase for complementation with the omega fragment in vivo. A construct consisting of Streptavadin-Linker-Alpha fragment was transformed into BL21(DE3) <i>E.coli</i> cells that naturally express the omega fragment of beta-galactosidase. Quadrant streak plate in the presence of X-gal produced dark blue colonies. These results illustrate alpha-omega complementation <i>in vivo</i>. In vivo complementation indicates the ability of the two fragments to fuse into a functional beta-galactosidase unit, indicating that the split beta-galactosidase reporter system module of the biosensor was constructed and can be implemented successfully.
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</p>
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<br />
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Etc….
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<div align="center">
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<div style="width:800px; padding:10px; border:2px solid gray; margin:0px; background-color:aliceblue; text-align:justify;">
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Notably, our data shows that the alpha fragment of beta-galactosidase was still able to complementarily bind to the omega fragment and produce a functional unit while linked to streptavidin, a toxic protein due to its high affinity towards biotin, an essential cofactor for fatty acid synthesis, valine synthesis, and gluconeogenesis. This indicates that the split beta-galactosidase reporter system can still be produced under harsh conditions and within a fusion protein construct.  This parallels the conditions that we expect our probe to mature in, given that the beta-galactosidase fragments will also be fused to topoisomerase, which is also a toxic protein that binds DNA. This provides a proof-of-concept for the DNA-based biosensor, given that both modules of the final biosensor design work as expected.
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</div>
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</div>
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<br />
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<h2>Current Research</h2>
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<p>
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Current testing with the split beta-galactosidase system includes time-interval testing of colorimetric response, including quantitative measurements of beta-galactosidase concentration over time, omega fragment negative control testing, and in vitro testing of the alpha and omega fragments linked to streptavadin and Magainin.
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</p>
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</body>
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</html>

Latest revision as of 05:53, 26 October 2012

Data

Topoisomerase-based DNA Biosensor

Data For Our New Favorite Parts

D168A Double Cysteine Mutant of Smallpox Topoisomerase, BBa_K891234

This mutant version of topoisomerase recognizes the YCCTT motif in dsDNA. It cleaves after the last T in this motif, making a single stranded nick, and covalently binds to the 3' phosphate on that thymine.

GFPT1, BBa_K891000

This part should be paired with GFPT2. This part codes for a 20bp sequence that is complementary to a portion of the genomic GFP coding sequence in E.coli Keio strains.

GFPT2, BBa_K891999

This part should be paired with GFPT1. This part codes for a 20bp sequence that is complementary to a portion of the genomic GFP coding sequence that comes after the GFPT1 binding site in E.coli Keio strains.


Split Beta-Galactosidase Reporter System

Tested alpha fragment of beta-galactosidase for complementation with the omega fragment in vivo. A construct consisting of Streptavadin-Linker-Alpha fragment was transformed into BL21(DE3) E.coli cells that naturally express the omega fragment of beta-galactosidase. Quadrant streak plate in the presence of X-gal produced dark blue colonies. These results illustrate alpha-omega complementation in vivo. In vivo complementation indicates the ability of the two fragments to fuse into a functional beta-galactosidase unit, indicating that the split beta-galactosidase reporter system module of the biosensor was constructed and can be implemented successfully.


Notably, our data shows that the alpha fragment of beta-galactosidase was still able to complementarily bind to the omega fragment and produce a functional unit while linked to streptavidin, a toxic protein due to its high affinity towards biotin, an essential cofactor for fatty acid synthesis, valine synthesis, and gluconeogenesis. This indicates that the split beta-galactosidase reporter system can still be produced under harsh conditions and within a fusion protein construct. This parallels the conditions that we expect our probe to mature in, given that the beta-galactosidase fragments will also be fused to topoisomerase, which is also a toxic protein that binds DNA. This provides a proof-of-concept for the DNA-based biosensor, given that both modules of the final biosensor design work as expected.

Current Research

Current testing with the split beta-galactosidase system includes time-interval testing of colorimetric response, including quantitative measurements of beta-galactosidase concentration over time, omega fragment negative control testing, and in vitro testing of the alpha and omega fragments linked to streptavadin and Magainin.

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