Team:UIUC-Illinois/Project/Future/Scaffold
From 2012.igem.org
Line 55: | Line 55: | ||
<br/> | <br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/3/3e/D0_Scaffold_From_Paper.png" height=100% width=100%><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/3/3e/D0_Scaffold_From_Paper.png" height=100% width=100%><br/><br/></center> |
<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/> | ||
Line 61: | Line 61: | ||
<br/><br/> | <br/><br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/2/2f/D0_Pam_Silver_DNA_Sequence.png"><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/2/2f/D0_Pam_Silver_DNA_Sequence.png"><br/></center><br/> |
<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. | ||
Line 67: | Line 67: | ||
<br/><br/> | <br/><br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/3/3e/D0_Scaffold.png" height=100% width=100%><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/3/3e/D0_Scaffold.png" height=100% width=100%><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. | ||
Line 73: | Line 73: | ||
<br/><br/> | <br/><br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/5/5c/Modified_d0_Sequence_with_PUF_Binding_Sites_--3.png"><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/5/5c/Modified_d0_Sequence_with_PUF_Binding_Sites_--3.png"><br/></center><br/> |
<b>Fig. 3.</b> | <b>Fig. 3.</b> | ||
Line 80: | Line 80: | ||
<br/><br/> | <br/><br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/d/d1/Freiburg_RNA_Tools_Website_Modifiedd_d0_-1.png" height=100% width=100%><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/d/d1/Freiburg_RNA_Tools_Website_Modifiedd_d0_-1.png" height=100% width=100%></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. | ||
Line 88: | Line 88: | ||
- | <img src="https://static.igem.org/mediawiki/2012/d/d2/IDT_miniGene_Scaffold_-1_--4.png"><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/d/d2/IDT_miniGene_Scaffold_-1_--4.png"></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. | ||
Line 96: | Line 96: | ||
- | <img src="https://static.igem.org/mediawiki/2012/c/c7/Freiburg_RNA_Tools_Website_Modifiedd_d0_-2.png" height=100% width=100%><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/c/c7/Freiburg_RNA_Tools_Website_Modifiedd_d0_-2.png" height=100% width=100%></center><br/><br/> |
<b>Fig. 7.</b> | <b>Fig. 7.</b> | ||
Line 102: | Line 102: | ||
<br/><br/> | <br/><br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/0/03/Aptamer_Concept_third_last.png"><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/0/03/Aptamer_Concept_third_last.png"></center><br/><br/> |
<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. | ||
Line 108: | Line 108: | ||
<br/><br/> | <br/><br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/6/6b/Split-GFP_Binding_Assay_second_last.png"><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/6/6b/Split-GFP_Binding_Assay_second_last.png"></center><br/><br/> |
<b>Fig. 9.</b> | <b>Fig. 9.</b> | ||
Line 115: | Line 115: | ||
<br/><br/> | <br/><br/> | ||
- | <img src="https://static.igem.org/mediawiki/2012/2/25/UNC_PUF-PIN_Endonuclease_Assay_last.png"><br/> | + | <center><img src="https://static.igem.org/mediawiki/2012/2/25/UNC_PUF-PIN_Endonuclease_Assay_last.png"></center><br/><br/> |
<b>Fig. 10.</b> | <b>Fig. 10.</b> | ||
Revision as of 07:46, 3 October 2012