Team:UIUC-Illinois/Project/Future/Scaffold

From 2012.igem.org

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<b>Fig. 6.</b>
<b>Fig. 6.</b>
The resulting scaffold of the changed sequence.  
The resulting scaffold of the changed sequence.  
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<b>Fig. 4.</b>
<b>Fig. 4.</b>
The scaffold was further modified after research suggested that PUF binds best to nucleotides with an angle of curvature similar to its own of approximately 20o turn per repeat*. The hairpin loops were changed in order to accommodate this from 8 nucleotides to 18 nucleotides achieving a 20o turn per nucleotide effect. Sequences of the stem loop were further modified in order to keep GC content from being too high and to make a more stable structure. This DNA sequence was then synthesized through IDT’s miniGENE option.
The scaffold was further modified after research suggested that PUF binds best to nucleotides with an angle of curvature similar to its own of approximately 20o turn per repeat*. The hairpin loops were changed in order to accommodate this from 8 nucleotides to 18 nucleotides achieving a 20o turn per nucleotide effect. Sequences of the stem loop were further modified in order to keep GC content from being too high and to make a more stable structure. This DNA sequence was then synthesized through IDT’s miniGENE option.
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<b>Fig. 7.</b>
<b>Fig. 7.</b>
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<b>Fig. 8.</b>
<b>Fig. 8.</b>
The figure above shows an important theoretical concept of the RNA scaffold. Assuming that the RNA binding proteins bind specifically, a spatial control of “cargo” can be made to serve various functions. In our project, we envisioned enzymes of the piceatannol pathway to churn out product due to the spatial proximity of one enzyme next to another, yielding increased reaction kinetics. However, in order to prove that such a concept is possible, a few assays need to be done.  
The figure above shows an important theoretical concept of the RNA scaffold. Assuming that the RNA binding proteins bind specifically, a spatial control of “cargo” can be made to serve various functions. In our project, we envisioned enzymes of the piceatannol pathway to churn out product due to the spatial proximity of one enzyme next to another, yielding increased reaction kinetics. However, in order to prove that such a concept is possible, a few assays need to be done.  
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<center><img src="http://2012.igem.org/wiki/images/6/6b/Split-GFP_Binding_Assay_second_last.png" height=75% width=75%><br/></center><br/>
<b>Fig. 9.</b>
<b>Fig. 9.</b>
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<b>Fig. 10.</b>
<b>Fig. 10.</b>

Revision as of 07:56, 3 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.

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