Team:Arizona State/Data

From 2012.igem.org

(Difference between revisions)
 
(34 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:Arizona_State/Template:Header}}
{{:Team:Arizona_State/Template:Header}}
-
 
-
Note from Dr. Haynes: Hi Nisarg<br>
 
-
 
-
Please add an outline of the experiments-in-progress on the "Data" page. For instance…
 
-
 
-
 
-
Experiment 1<br>
 
-
Aim: To determine that the split Beta-gal part of the biosensor works<br>
 
-
Approach: Co-express different versions of the alpha and omega fragments in E. coli<br>
 
-
Expected Results: If it works as expected, alpha and omega pairs will give a blue phenotype, and single units (negative controls) will not<br>
 
-
Lead experimenter: Abhi<br>
 
-
Progress: (to be filled in by Abhi)
 
-
 
-
 
-
Experiment 2<br>
 
-
Aim: To quantitate Topo enzyme DNA nicking using a crazy sequencing approach<br>
 
-
 
-
Etc….
 
<html>
<html>
<body>
<body>
<h1>Data</h1>
<h1>Data</h1>
-
<h2>ssDNA Probe Design</h2>
+
<hr style="color: #800000; height:3px;" />
-
<h3>Topoisomerase-based DNA Biosensor</h3>
+
 
 +
<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>
-
insert diagram here
+
<a href="http://partsregistry.org/Part:BBa_K891234">D168A Double Cysteine Mutant of Smallpox Topoisomerase, BBa_K891234</a>
</p>
</p>
-
<h4>Data For Our New Favorite Parts</h4>
 
<p>
<p>
-
<a href="http://partsregistry.org/Part:BBa_K891234">D168A Double Cysteine Mutant of Smallpox Topoisomerase, BBa_K891234</a> - 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 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> - 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.
+
<a href="http://partsregistry.org/Part:BBa_K891000">GFPT1, BBa_K891000</a>
</p>
</p>
<p>
<p>
-
<a href="http://partsregistry.org/Part:BBa_K891999">GFPT2, BBa_K891999</a> - 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.
+
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>
-
<h2>DNA Biosensor</h2>
+
<p>
-
<h3>Split Beta-Galactosidase Complementation</h3>
+
<a href="http://partsregistry.org/Part:BBa_K891999">GFPT2, BBa_K891999</a>
 +
</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>
 +
<br />
 +
 
 +
<h2>Split Beta-Galactosidase Reporter System</h2>
 +
 
 +
<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>
 +
<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>
 +
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.
 +
</p>
 +
 
 +
</body>
 +
</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.

Retrieved from "http://2012.igem.org/Team:Arizona_State/Data"