Team:UIUC-Illinois/Project/Future/AssemblyLine

From 2012.igem.org

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<br/><br/> After gathering the four enzymes we would use in our assembly line, we began to tackle the simplest steps first. Since we planned to characterize BM3 while integrating it into the assembly line, we chose to begin testing its ability to synthesize piceatannol from resveratrol in vivo. We needed to discern whether piceatannol would be exported from the cell after production, so we devised an assay to differentiate piceatannol from resveratrol and other cellular products in cell growth medium. Using thin-layer liquid chromatography (TLC), we experimented with different procedures for separating control samples of resveratrol and piceatannol.  
<br/><br/> After gathering the four enzymes we would use in our assembly line, we began to tackle the simplest steps first. Since we planned to characterize BM3 while integrating it into the assembly line, we chose to begin testing its ability to synthesize piceatannol from resveratrol in vivo. We needed to discern whether piceatannol would be exported from the cell after production, so we devised an assay to differentiate piceatannol from resveratrol and other cellular products in cell growth medium. Using thin-layer liquid chromatography (TLC), we experimented with different procedures for separating control samples of resveratrol and piceatannol.  
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<b>Fig. 1.</b> From left to right, the plate lanes are marked Resveratrol, Piceatannol, Biofermentation (WT, 10, or 13), and Reference (containing both Resveratrol and Piceatannol)<br/>
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We were able to achieve separation of the controls on a normal phase TLC plate, but when we applied the concentrated supernatant of biofermentations of BM3 culture, we were unable to see piceatannol on the TLC plate. In the interest of the PUF project, we suspended work on this project but hope to work toward the realization of a enzymatic assembly line in the future.
We were able to achieve separation of the controls on a normal phase TLC plate, but when we applied the concentrated supernatant of biofermentations of BM3 culture, we were unable to see piceatannol on the TLC plate. In the interest of the PUF project, we suspended work on this project but hope to work toward the realization of a enzymatic assembly line in the future.

Revision as of 02:38, 4 October 2012

Header

Assembly

Enzymatic
Assembly Line

  • Overview
  • Design and Theory
  • Approach
  • Prospective
  • Enzymatic Assembly Line Overview



    Earlier this year, research at the Kee-Hong Kim lab of Purdue University had preliminary evidence showing that a trans-stilbene compound, Piceatannol, had an ability to inhibit the development of human adipose cells. The mechanism is based around the idea that Piceatannol interacts with a preadipocyte's (immature fat cell) insulin receptors in such a way that surpresses it's growth into a mature adipose cell. Piceatannol is a metabolite of resveratrol, a compoud currently under investigation for possible anti-cancer properites. Piceatannol differs from resveratrol by one hydroxyl group on one of the aromatic rings.

    Piceatannol is currently very costly to synthesize. On the advent of such a discovery, we felt that if we were to engineer a pathway to optimize the production of Piceatannol from cheaper substrates through the utilization of our PUF and RNA scaffold projects, we could show the versatility of our PUF toolkit working with an RNA scaffold.

    However, before researchers can begin using our PUF toolkit to produce piceatannol in vivo, we took steps to acquire and characterize the necessary genes for our: