Team:UIUC-Illinois/Project/Future/Scaffold

From 2012.igem.org

(Difference between revisions)
Line 56: Line 56:
<center><img src="http://2012.igem.org/wiki/images/3/3e/D0_Scaffold_From_Paper.png" height=100% width=100%><br/><br/></center>
<center><img src="http://2012.igem.org/wiki/images/3/3e/D0_Scaffold_From_Paper.png" height=100% width=100%><br/><br/></center>
-
<b>Fig. 1.</b><sup>1</sup> The design of our RNA scaffold was based upon a scaffold created by the <a href="http://openwetware.org/wiki/User:PamSilver">Pam Silver</a> research lab at Harvard. This group was the only one to develop such a construct (pictured above) and prove its effectiveness so it was built upon in order to serve as the application for our own RNA binding proteins. <br/><br/>
+
<b>Fig. 1.</b><sup>[1]</sup> The design of our RNA scaffold was based upon a scaffold created by the <a href="http://openwetware.org/wiki/User:PamSilver">Pam Silver</a> research lab at Harvard. This group was the only one to develop such a construct (pictured above) and prove its effectiveness so it was built upon in order to serve as the application for our own RNA binding proteins. <br/><br/>
The <a href="http://www.sciencemag.org/content/333/6041/470.abstract">d0 scaffold</a> which is shown above has two hairpin loops with binding sites for two distinct RNA binding proteins, MS2 and PP7. Although the Silver group developed even more complicated scaffolds, we decided to work with the simplest (d0) as a way to prove not only that PUF can bind specifically, but that the scaffold can be used for efficient production of compounds in bacterial cells.
The <a href="http://www.sciencemag.org/content/333/6041/470.abstract">d0 scaffold</a> which is shown above has two hairpin loops with binding sites for two distinct RNA binding proteins, MS2 and PP7. Although the Silver group developed even more complicated scaffolds, we decided to work with the simplest (d0) as a way to prove not only that PUF can bind specifically, but that the scaffold can be used for efficient production of compounds in bacterial cells.

Revision as of 03:30, 4 October 2012

Header

Scaffold

RNA Scaffold

  • Overview
  • RNA Scaffold Design
  • PUF Tethering Design
  • RNA Scaffold Overview


    In order to provide a direct application for the RNA binding abilities of PUF, an RNA scaffold was designed with the idea of serving as a platform for an enzyme conveyor belt. The array of enzymatic pathways which could be enhanced by a scaffold are numerous, though, we projected to increase efficiency and production of a resveratrol derivative called piceatannol.

    The project consists of a couple parts, each a proof of concept and build-up of previous ones. The start of the project consisted of designing an RNA scaffold which is best tailored to PUF binding in a spatially specific manner. Once the scaffold was designed, synthesized, and purified it was important to show not only that PUF can bind specifically to its designated sites, but that the scaffold can support a concept such as a biological conveyor belt.

    One assay which was designed to prove this was incubation of the RNA scaffold with non-specific endonucleases bound to PUF. The length of digested RNA parts would prove that PUF was binding specifically and appropriately to the designated sequences. Another assay would include tethering a split-fluorescent protein to wild-type and mutant PUF. An in-vitro gel-shift assay, or EMSA, would once again prove that PUF is binding the the appropriate sites. More importantly, an in-vivo experiment which shows fluorescence with presence of the scaffold and darkness without the scaffold would prove efficient enzymatic pathways could be achieved.

    Retrieved from "http://2012.igem.org/Team:UIUC-Illinois/Project/Future/Scaffold"