Freiburg07/report Ca sensor
From 2007.igem.org
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
| + | |||
| + | Split enzymes:<br> | ||
| + | Some enzymes can be divided into two separate parts that won´t show any activity until both parts are physically brought together again;<br> | ||
| + | this circumstance allows enzyme-activity-assays.<br> | ||
| + | As those assays were already being used in our lab before, we could access plasmids containing enzyme-halfs of beta-lactamase and Dihydrofolatereductase (DHFR).<br> | ||
| + | |||
| + | The idea:<br> | ||
| + | Was to attach those enzyme-halfs to several trigger-proteins (here: calmoduline, which provides a strong conformational change upon binding calcine)<br> | ||
| + | by cloning each one of the enzyme-halfs and the trigger-protein together in one plasmid.<br> | ||
| + | This plasmid would then contain the code for the whole "protein-machinery", allowing in vivo-tests as well as expression and purification of the engineered protein, an on/off-switchable enzyme.<br> | ||
<h2>Materials and Methods</h2> | <h2>Materials and Methods</h2> | ||
Revision as of 11:18, 23 October 2007
Contents |
A single-protein calcium ion sensor mediating cell survival
iGEM team Freiburg
Introduction
Split enzymes:
Some enzymes can be divided into two separate parts that won´t show any activity until both parts are physically brought together again;
this circumstance allows enzyme-activity-assays.
As those assays were already being used in our lab before, we could access plasmids containing enzyme-halfs of beta-lactamase and Dihydrofolatereductase (DHFR).
The idea:
Was to attach those enzyme-halfs to several trigger-proteins (here: calmoduline, which provides a strong conformational change upon binding calcine)
by cloning each one of the enzyme-halfs and the trigger-protein together in one plasmid.
This plasmid would then contain the code for the whole "protein-machinery", allowing in vivo-tests as well as expression and purification of the engineered protein, an on/off-switchable enzyme.
