Team:MIT/ResultsActuation

From 2012.igem.org

Revision as of 23:14, 2 October 2012 by Kipnissn (Talk | contribs)

DEPRECATED. DO NOT USE OR EDIT. If a page uses this template, relink with MIT-results2.

Decoys and Tough Decoys (TuDs)

We wanted something that would provide a tight double repression system with a very distinct change between on and off. The TuDs and Decoys design were originally inspired by the “Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells,” paper. We copied their designs and wanted to reproduce the results in our lab. To do so, we ordered TuDs and decoys both with and without bulges. The bulges are designed to disrupt RISC complex activity; something which degrades short RNA like our decoys in the cell.
Sources: http://nar.oxfordjournals.org/content/early/2009/02/17/nar.gkp040.abstract
http://www.ncbi.nlm.nih.gov/pubmed/9695408

FF1 Knockdown

In order to transcribe microRNAs, we first needed to characterize the U6 promoter. To do so, we utilized a system that constitutively expresses eYFP with a microRNA site attached. Adding the U6:miRNA, we see knockdown of the gene. By varying molar ratios of miRNA to fluorescent signal, we can calibrate the strength of the U6 promoter. Another advantage to this system is that it can serve as an actuation method. A miRNA targeting a gene of interest could be the output to a RNA circuit.

Legend: TO BE FILLED IN 100,000 HEK293 Cells were transfected with varying molar ratios of U6-tet-o:mirFF1 to Hef1a:eYFP 4xFF1, and 1:1 molar ratio of Hef1a:eYFP 4xFF1 and Hef1a:TagBFP (as a transfection marker), standardized to a total of 500ng plasmid DNA using 1.65 uL of lipofectamine 2000. As we increase the ratio of mirFF1 from 1/4x to 8x, there is a decrease in the yellow fluorescent signal, indicating gene knockdown. The histograms show the population of cells shifting from yellow towards the non fluorescent region.

Retrieved from "http://2012.igem.org/Team:MIT/ResultsActuation"