Difference between revisions of "Team:Technion Israel/Project/Results"
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
| + | <h2>Expression</h2> | ||
| + | <p>After successful overexpression of the 3ɑ-HSD enzyme under pT7 promoter (<a href="http://parts.igem.org/Part:BBa_K1674002" target="_blank">BBa_K1674002</a>), we conducted a series of experiments based on the 3ɑ-HSD activity measurement protocol, where we measured NADPH fluorescence over time added to E.coli lysates. Every experiment helped us understand a different aspect of the dihydrotestosterone (DHT) reduction reaction using our clones, and characterize the plasmid.</p> | ||
| + | |||
| + | <h3>Enzymatic activity as a function of DHT concentration</h3> | ||
| + | <p>To get a basic idea of the kinetics of 3ɑ-HSD enzymatic reaction, we first wanted to examine the effect of increase in initial substrate concentration (DHT).</p> | ||
| + | <p>We sonicated BL21 cells after two hours induction with IPTG, added 150uM NADPH to the lysates in a 96-well plate and inserted into plate reader at 37℃ for 30 minutes for stabilization.</p> | ||
| + | <h2>Secretion</h2> | ||
| + | <h2>Cofactor</h2> | ||
| + | <h2>Integrative experiments and shelf-life</h2> | ||
Revision as of 00:25, 18 September 2015
Results
Introduction
Expression
After successful overexpression of the 3ɑ-HSD enzyme under pT7 promoter (BBa_K1674002), we conducted a series of experiments based on the 3ɑ-HSD activity measurement protocol, where we measured NADPH fluorescence over time added to E.coli lysates. Every experiment helped us understand a different aspect of the dihydrotestosterone (DHT) reduction reaction using our clones, and characterize the plasmid.
Enzymatic activity as a function of DHT concentration
To get a basic idea of the kinetics of 3ɑ-HSD enzymatic reaction, we first wanted to examine the effect of increase in initial substrate concentration (DHT).
We sonicated BL21 cells after two hours induction with IPTG, added 150uM NADPH to the lysates in a 96-well plate and inserted into plate reader at 37℃ for 30 minutes for stabilization.