Team:UIUC-Illinois/Project/Future/Scaffold
From 2012.igem.org
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<b>Fig. 1.</b> 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> 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. | ||
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- | <center><img src="https://static.igem.org/mediawiki/2012/2/2f/D0_Pam_Silver_DNA_Sequence.png" width=100%><br/></ | + | <center><img src="https://static.igem.org/mediawiki/2012/2/2f/D0_Pam_Silver_DNA_Sequence.png" width=100%><br/><br/></center> |
<b>Fig. 2.</b> | <b>Fig. 2.</b> | ||
The sequence in Fig. 2 is the DNA sequence coding for the d0 scaffold. The first highlighted potion is the T7 promoter followed by the MS2 binding site. The next highlighted region shows the PP7 binding site followed by the T7 terminator. | The sequence in Fig. 2 is the DNA sequence coding for the d0 scaffold. The first highlighted potion is the T7 promoter followed by the MS2 binding site. The next highlighted region shows the PP7 binding site followed by the T7 terminator. | ||
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- | <center><img src="https://static.igem.org/mediawiki/2012/3/3e/D0_Scaffold.png" height= | + | <center><img src="https://static.igem.org/mediawiki/2012/3/3e/D0_Scaffold.png" height=50% width=50%><br/></center><br/> |
<b>Fig. 5.</b> | <b>Fig. 5.</b> | ||
The corresponding image of the d0 RNA secondary structure as predicted by the <a href="http://rna.informatik.uni-freiburg.de:8080/LocARNA.jsp">LocARNA tool of Freiburg RNA Tools</a>. The software accurately depicts what the d0 structure looks like in the literature, therefore it was a reliable program which could be used to modify and visualize RNA secondary structures. | The corresponding image of the d0 RNA secondary structure as predicted by the <a href="http://rna.informatik.uni-freiburg.de:8080/LocARNA.jsp">LocARNA tool of Freiburg RNA Tools</a>. The software accurately depicts what the d0 structure looks like in the literature, therefore it was a reliable program which could be used to modify and visualize RNA secondary structures. | ||
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<center><img src="https://static.igem.org/mediawiki/2012/5/5c/Modified_d0_Sequence_with_PUF_Binding_Sites_--3.png" width=100% ><br/></center><br/> | <center><img src="https://static.igem.org/mediawiki/2012/5/5c/Modified_d0_Sequence_with_PUF_Binding_Sites_--3.png" width=100% ><br/></center><br/> | ||
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Modifications to the d0 sequence were made by replacing the PP7 and MS2 binding sites with WT PUF-PIN and 6-2/7-2 PUF-PIN binding sites. | Modifications to the d0 sequence were made by replacing the PP7 and MS2 binding sites with WT PUF-PIN and 6-2/7-2 PUF-PIN binding sites. | ||
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- | <center><img src="https://static.igem.org/mediawiki/2012/d/d1/Freiburg_RNA_Tools_Website_Modifiedd_d0_-1.png" height= | + | <center><img src="https://static.igem.org/mediawiki/2012/d/d1/Freiburg_RNA_Tools_Website_Modifiedd_d0_-1.png" height=50% width=50%></center><br/><br/> |
<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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- | <center><img src="https://static.igem.org/mediawiki/2012/d/d2/IDT_miniGene_Scaffold_-1_--4.png" width= | + | <center><img src="https://static.igem.org/mediawiki/2012/d/d2/IDT_miniGene_Scaffold_-1_--4.png" height=50% width=50%></center><br/><br/> |
<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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The final structure of the scaffold used in the project. | The final structure of the scaffold used in the project. | ||
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<center><img src="https://static.igem.org/mediawiki/2012/0/03/Aptamer_Concept_third_last.png"></center><br/><br/> | <center><img src="https://static.igem.org/mediawiki/2012/0/03/Aptamer_Concept_third_last.png"></center><br/><br/> | ||
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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="https://static.igem.org/mediawiki/2012/6/6b/Split-GFP_Binding_Assay_second_last.png"></center><br/><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/6/6b/Split-GFP_Binding_Assay_second_last.png" height=50% width=50%></center><br/><br/> |
<b>Fig. 9.</b> | <b>Fig. 9.</b> | ||
A gel-shift in-vitro assay as the one pictured would properly demonstrate that distinct RNA binding proteins are binding to the scaffold and thus proper scaffold functioning. With addition of the scaffold, each separate binding protein causes a change in overall size resulting in a different band from each protein by itself. Due to assay shown being ran on a native gel, secondary structures and changes in electronegative affinities might explain the unusual effect of larger constructs being localized further down the gel. | A gel-shift in-vitro assay as the one pictured would properly demonstrate that distinct RNA binding proteins are binding to the scaffold and thus proper scaffold functioning. With addition of the scaffold, each separate binding protein causes a change in overall size resulting in a different band from each protein by itself. Due to assay shown being ran on a native gel, secondary structures and changes in electronegative affinities might explain the unusual effect of larger constructs being localized further down the gel. | ||
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- | <center><img src="https://static.igem.org/mediawiki/2012/2/25/UNC_PUF-PIN_Endonuclease_Assay_last.png" height= | + | <center><img src="https://static.igem.org/mediawiki/2012/2/25/UNC_PUF-PIN_Endonuclease_Assay_last.png" height=30% width=30%></center><br/><br/> |
<b>Fig. 10.</b> | <b>Fig. 10.</b> | ||
This figure is shown from the Prof. Wang’s lab in UNC which characterized PUF-PIN (PIN being a non-specific endonuclease) functioning at various pH levels. A similar assay with our scaffold would show specific binding of PUF to its destined sites on the scaffold due to the presence of RNA fragments of expected lengths. | This figure is shown from the Prof. Wang’s lab in UNC which characterized PUF-PIN (PIN being a non-specific endonuclease) functioning at various pH levels. A similar assay with our scaffold would show specific binding of PUF to its destined sites on the scaffold due to the presence of RNA fragments of expected lengths. | ||
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</div> | </div> |
Revision as of 07:52, 3 October 2012