Difference between revisions of "Team:Paris Bettencourt/Production"
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<p>Cell-free extracts were assayed for the presence of StarCore proteins by a variety of methods, described in the Characterization page. Unfortunately, none of these assayes produced evidence of successful protein expression and this despite our tests with the same constructs encoding eGFP instead of our StarCore.</p> | <p>Cell-free extracts were assayed for the presence of StarCore proteins by a variety of methods, described in the Characterization page. Unfortunately, none of these assayes produced evidence of successful protein expression and this despite our tests with the same constructs encoding eGFP instead of our StarCore.</p> | ||
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| + | <div class="textbody table tr td"> | ||
| + | <table boarder=5 cellpassing=0 cellpadding="12" width="500"> | ||
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| + | <th allign="center">Box 1: Why are StarCore Proteins Difficult to Express?</th> | ||
| + | </tr> | ||
| + | <tr height=100> | ||
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| + | <p>Although our cell-free mix performed well for expression of GFP controls, we were not able to achieve sufficient yields of StarCore proteins. It is clear that AMPs present special challenges to cell-free expression. Here are some reasons we came up with for why StarCores might be difficult to express with high yield.</p> | ||
| + | <br><li>Unbalanced amino acid usage, with a high density of lysine and arginine residues.</li></br> | ||
| + | <li>High density of positive charges, leading to misfolding and aggregation.</li></br> | ||
| + | <li>Direct toxicity to cell components caused by AMP activity.</li> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
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Revision as of 13:29, 17 October 2018
Production
In the Design section, we designed StarCore sequences as compound BioBricks fusing an AMP sequence to a multimeric core. In this section, we use cell-free expression to produce StarCore proteins.
Cell-free expression allows us to bypass the toxic effect that our living cells would have if they produced our StarCore for hours but also allows us to have high throughput expression and to quickly screen many compounds.
Results
Expression of the StarCore Fusion Proteins
StarCore fusion proteins were expressed using the myTXTL Sigma 70 Master Mix Kit, generously provided by our team sponsor, Arbor Biosciences. As an expression vector, we used pACYCDuet-1 from Novagen. This vector is widely used for protein production in strains of E. coli that express T7 polymerase such as BL21 (DE3). It contains a T7 promoter upstream of a strong RBS and a lac operator, allowing IPTG-controllable protein expression.
To express from the T7 promoter, it was necessary to first produce T7 polymerase in the cell-free extract. For this purpose, we used the plasmid P70a-T7rnap, supplied by the manufacturer. We also included 100 uM IPTG in the master mix, to relieve lac repression.
Cell-free extracts were assayed for the presence of StarCore proteins by a variety of methods, described in the Characterization page. Unfortunately, none of these assayes produced evidence of successful protein expression and this despite our tests with the same constructs encoding eGFP instead of our StarCore.
| Box 1: Why are StarCore Proteins Difficult to Express? |
|---|
|
Although our cell-free mix performed well for expression of GFP controls, we were not able to achieve sufficient yields of StarCore proteins. It is clear that AMPs present special challenges to cell-free expression. Here are some reasons we came up with for why StarCores might be difficult to express with high yield. |
paris-bettencourt-2018@cri-paris.org 
