Team:UIUC-Illinois/Project/Future/Scaffold

From 2012.igem.org

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<center><img src="http://2012.igem.org/wiki/images/d/d2/IDT_miniGene_Scaffold_-1_--4.png" width=100%><br/></center><br/>
<center><img src="http://2012.igem.org/wiki/images/d/d2/IDT_miniGene_Scaffold_-1_--4.png" width=100%><br/></center><br/>
<b>Fig. 6.</b>
<b>Fig. 6.</b>
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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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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 20<sup>o</sup> turn per repeat <sup>[3]</sup>. The hairpin loops were changed in order to accommodate this from 8 nucleotides to 18 nucleotides achieving a 20<sup>o</sup> 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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<u>References</u>:<br/>
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<u>References</u>:<br/><br/>
[1]:<br/>
[1]:<br/>
Organization of Intracellular Reactions with Rationally Designed RNA Assemblies<br/>
Organization of Intracellular Reactions with Rationally Designed RNA Assemblies<br/>
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Engineering RNA Endonucleases with Customized Sequence Specificities<br/>
Engineering RNA Endonucleases with Customized Sequence Specificities<br/>
Rajarshi Choudhury, Daniel Dominguez, Yang Wang and Zefeng Wang<br/>
Rajarshi Choudhury, Daniel Dominguez, Yang Wang and Zefeng Wang<br/>
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Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, April 11, 2012<br/><br/>
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Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, April 11, 2012<br/>
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[3]: <br/>Yeming Wang,  Laura Opperman,  Marvin Wickens,  and Traci M. Tanaka Hall. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein. PNAS 2009 ; published ahead of print November 9, 2009,doi:10.1073/pnas.0812076106<br/>
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<center><img src="http://2012.igem.org/wiki/images/f/fa/EGFP_Concept_2.png" ><br/></center><br/>
<center><img src="http://2012.igem.org/wiki/images/f/fa/EGFP_Concept_2.png" ><br/></center><br/>
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<b>Fig. 1.</b>
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<b>Fig. 1.</b><sup>[1]</sup>
An example of a conclusive experiment which would prove that spatial control of enzymes is possible is depicted above. With addition of split fluorescent parts to each separate RNA binding protein observed fluorescence with expression of the scaffold is expected. This result would prove spatial control of enzymes is possible and a desired effect, such as an enzyme conveyer belt, is achievable.
An example of a conclusive experiment which would prove that spatial control of enzymes is possible is depicted above. With addition of split fluorescent parts to each separate RNA binding protein observed fluorescence with expression of the scaffold is expected. This result would prove spatial control of enzymes is possible and a desired effect, such as an enzyme conveyer belt, is achievable.
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<center><img src="http://2012.igem.org/wiki/images/c/c4/Flourescent_Microscopy_Assay_3.png" height=100% width=100%><br/></center><br/>
<center><img src="http://2012.igem.org/wiki/images/c/c4/Flourescent_Microscopy_Assay_3.png" height=100% width=100%><br/></center><br/>
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<b>Fig. 2.</b>
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<b>Fig. 2.</b><sup>[1]</sup>
Fluorescence microscopy would show that activity of the fluorescent protein only occurs with addition of the RNA scaffold. The scaffold brings the split-fluorescent parts to close proximity resulting in a drastic effect such as an almost 100 fold in fluorescence.
Fluorescence microscopy would show that activity of the fluorescent protein only occurs with addition of the RNA scaffold. The scaffold brings the split-fluorescent parts to close proximity resulting in a drastic effect such as an almost 100 fold in fluorescence.
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<b>Fig. 3.</b>
<b>Fig. 3.</b>
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In order to tether split-fluorescent proteins to PUF, primer extension was used with BioBricks from the Parts Registry. Specifically, parts <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K157005">BBa_K157005</a> (Split-Cerulean-CFP) and <a href="http://partsregistry.org/Part:BBa_K157006">BBa_K157006</a> (Split-Cerulean-nCFP) were worked with after the 2010 Slovenia iGEM team showed conclusive results testing them. Linker sequences used in literature which bound PUF to endonucleases were included in the primer sequences to create the most optimal construct*. Primes on the outside of the gene parts included restriction enzymes compatible with standard assembly methods for future biobricking work.  
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In order to tether split-fluorescent proteins to PUF, primer extension was used with BioBricks from the Parts Registry. Specifically, parts <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K157005">BBa_K157005</a> (Split-Cerulean-CFP) and <a href="http://partsregistry.org/Part:BBa_K157006">BBa_K157006</a> (Split-Cerulean-nCFP) were worked with after the 2010 Slovenia iGEM team showed conclusive results testing them. Linker sequences used in literature which bound PUF to endonucleases were included in the primer sequences to create the most optimal construct <sup>[2]</sup>. Primes on the outside of the gene parts included restriction enzymes compatible with standard assembly methods for future biobricking work.  
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<br/><br/>
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<u>References</u>:<br/><br/>
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[1]:<br/>
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Organization of Intracellular Reactions with Rationally Designed RNA Assemblies<br/>
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Camille J. Delebecque,  Ariel B. Lindner,  Pamela A. Silver,  and Faisal A. Aldaye <br/>
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Science 22 July 2011: 333 (6041), 470-474.Published online 23 June 2011 [DOI:10.1126/science.1206938]<br/><br/>
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[2]:<br/>
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Engineering RNA Endonucleases with Customized Sequence Specificities
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Rajarshi Choudhury, Daniel Dominguez, Yang Wang and Zefeng Wang
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Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, April 11, 2012
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Latest revision as of 04:14, 26 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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