Team:Uppsala University/Translational

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Revision as of 20:51, 25 September 2012

Team Uppsala University – iGEM 2012

Silencing Truncated AAC(6') by use of fluorescent reporter

In order to find candidates for conditional gene silencing, we used a method already described in [1] and adapted it to a modular biobrick RFC10 system. Briefly, the method goes like this:

Express a chimeric gene - The gene of interest translationally linked to a flourescent reporter
Express a library of small RNA's with a suitable stabilizing scaffold in the same strain as the reporter construct
Screen for lower flouresence as a result of downregulation of the reporter

Mechanism of downregulation

The general system for downregulation is illustrated above.
Stabilizing scaffold
In order to retain transcribed sRNA in E.coli and avoid degradation of the sRNA, a stabilizing scaffold can be linked to the domain which is hypothesized to interact with the domain of interest.
Hfq Protein
The Hfq Protein is a natively expressed protein which interacts with the scaffold. A deletion study of the Hfq protein resulted in total loss of regulation for many sRNAs [2].

References

[1] Sharma, V., Yamamura, A., Yokobayashi, Y., 2012. Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli. ACS Synth. Biol. 1, 6–13. [2] Holmqvist, E., Unoson, C., Reimegård, J., Wagner, E.G.H., 2012. A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp. Molecular Microbiology 84, 414–427.

@@ Line 43: / Line 43: @@
 <ul>
-<li>Express a chimeric gene - The gene interest linked to a reporter in a strain</li>
+<li>Express a chimeric gene - The gene of interest translationally linked to a flourescent reporter</li>
-<li>Express a library of small RNA's with a suitable stabilizing scaffold in the same strain</li>
+<li>Express a library of small RNA's with a suitable stabilizing scaffold in the same strain as the reporter construct
-<li>Screen for regulation of the reporter</li>
+</li>
+<li>Screen for lower flouresence as a result of downregulation of the reporter</li>
 </UL>
 </p>
 </td>
@@ Line 117: / Line 117: @@
 [1] Sharma, V., Yamamura, A., Yokobayashi, Y., 2012. Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli. ACS Synth. Biol. 1, 6–13.
+[2] Holmqvist, E., Unoson, C., Reimegård, J., Wagner, E.G.H., 2012. A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp. Molecular Microbiology 84, 414–427.