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Team:Arizona State/Data
From 2012.igem.org
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<html> | <html> | ||
<body> | <body> | ||
<h1>Data</h1> | <h1>Data</h1> | ||
| - | < | + | <hr style="color: #800000; height:3px;" /> |
| - | < | + | |
| + | <h2>Topoisomerase-based DNA Biosensor</h2> | ||
| + | |||
| + | <div align="center"> | ||
| + | <img src="https://static.igem.org/mediawiki/2012/8/8f/TopoDiagram.png" width="800" height="500" /> | ||
| + | </div> | ||
| + | |||
| + | <h3>Data For Our New Favorite Parts</h3> | ||
<p> | <p> | ||
| - | + | <a href="http://partsregistry.org/Part:BBa_K891234">D168A Double Cysteine Mutant of Smallpox Topoisomerase, BBa_K891234</a> | |
</p> | </p> | ||
| - | |||
<p> | <p> | ||
| - | + | 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. | |
</p> | </p> | ||
<p> | <p> | ||
| - | <a href="http://partsregistry.org/Part:BBa_K891000">GFPT1, BBa_K891000</a> | + | <a href="http://partsregistry.org/Part:BBa_K891000">GFPT1, BBa_K891000</a> |
</p> | </p> | ||
<p> | <p> | ||
| - | + | 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. | |
</p> | </p> | ||
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<p> | <p> | ||
| - | + | <a href="http://partsregistry.org/Part:BBa_K891999">GFPT2, BBa_K891999</a> | |
</p> | </p> | ||
| - | |||
<p> | <p> | ||
| - | < | + | 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. |
</p> | </p> | ||
| - | < | + | <br /> |
| + | |||
| + | <h2>Split Beta-Galactosidase Reporter System</h2> | ||
| + | |||
<p> | <p> | ||
| - | < | + | 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. |
</p> | </p> | ||
| - | < | + | <br /> |
| + | |||
| + | <div align="center"> | ||
| + | <div style="width:800px; padding:10px; border:2px solid gray; margin:0px; background-color:aliceblue; text-align:justify;"> | ||
| + | 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. | ||
| + | </div> | ||
| + | </div> | ||
| + | <br /> | ||
| + | |||
| + | |||
| + | <h2>Current Research</h2> | ||
| + | |||
<p> | <p> | ||
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. | 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. | ||
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.
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.
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.
