Difference between revisions of "Part:BBa K313010"
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Please see [http://2010.igem.org/Team:UT-Tokyo/Sudoku_assay_MS2  Phage MS2] assay page.<br/>
 
Please see [http://2010.igem.org/Team:UT-Tokyo/Sudoku_assay_MS2  Phage MS2] assay page.<br/>
  
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__NOTOC__
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We are SYSU-CHINA 2021 and this year we use circRNA to colocalize enzymes, which are fused to RNA binding proteins(RBP). We choose MS2 coat protein and PP7 coat protein as our RBP,and their aptamer on circRNA.For contribution, we predict its 2D and 3D structure,to present a visual impression.
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<br>Group: SYSU-CHINA 2021
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<br>Author: Zhao bingnan
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<br>Summary: the 2D and 3D sturcture and application of MS2 aptamer
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<br>resource:2018_Book_VirusProteinAndNucleoproteinCo,Organization of Intracellular Reactions with Rationally Designed RNA <br>Assemblies
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<br>Uploads: MS2 aptamer on the circRNA scaffold.png;SYSU-CHINA circRNA-1.png;SYSU-CHINA circRNA-2.png;The principle of spite EGFP.png;MS2 binding mechanism.png
  
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===Applications of BBa_K313010===
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We are SYSU-CHINA 2021 and this year we use circRNA to colocalize enzymes, which are fused to RNA binding proteins(RBP). We choose MS2 coat protein and PP7 coat protein as our RBP,and their aptamer on circRNA.For contribution, we predict its 2D and 3D structure,to present a visual impression.
  
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<!-- this is a RBS of MS2
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===2D stucture===
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===Usage and Biology===
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<!-- DON'T DELETE --><partinfo>BBa_K313010 StartReviews</partinfo>
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{|width='80%' style='border:1px solid gray'
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|-
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<partinfo>BBa_K313010 AddReview number</partinfo>
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<I>Username</I>
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experimental data
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|};
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<br>We use RNAfold and StructureEditor to predict 2D structure of our circRNA, which contains MS2 aptamer.The results are below:
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[[Image:MS2_aptamer_on_the_circRNA_scaffold.png ‎ | border | center | 300px]]
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<font size="1"><center>Figure 1.2D stucture predicted by RNAfold</center></font><br/>
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[[Image:SYSU-CHINA_circRNA-1.png ‎ | border | center | 300px]]
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<font size="1"><center>Figure 2.2D stucture predicted by StructureEditor</center></font><br/>
  
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===3D stucture===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K313010 SequenceAndFeatures</partinfo>
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|-
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<partinfo>BBa_K313010 AddReview number</partinfo>
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<I>Username</I>
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experimental data
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|};
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<!-- End of the user review template -->
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<br>We use this website:http://biophy.hust.edu.cn/new/3dRNA/create, to predict the 3D structure of our circRNA, which contains MS2 aptamer.The result lists below:
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<br>[[image:SYSU-CHINA circRNA-2.png |border|center|300px]]
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<br><font size="1"><center>Figure 3. 3D stucture of MS2 aptamer predicted by Xiaolab</center></font>
  
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===MS2 binding mechanism===
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<!-- DON'T DELETE --><partinfo>BBa_K313010 StartReviews</partinfo>
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{|width='80%' style='border:1px solid gray'
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|-
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<partinfo>BBa_K313010 AddReview number</partinfo>
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<I>Username</I>
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|width='60%' valign='top'|
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experimental data
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|};
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<br>[[Image:MS2 binding mechanism.png  | border | center | 300px]]
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<br><center><font size="1">Figure 4. MS2 binding mechanism</font></center>
  
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<!-- Uncomment this to enable Functional Parameter display
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<br>
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===Functional Parameters===
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===split EGFP===
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<partinfo>BBa_K313010 parameters</partinfo>
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<!-- Template for a user review
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{|width='80%' style='border:1px solid gray'
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<partinfo>BBa_K313010 AddReview number</partinfo>
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<I>Username</I>
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|width='60%' valign='top'|
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experimental data
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|};
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<br>We are SYSU-CHINA,and this year we use this part as MS2 coat protein's aptamer to do a split EGFP experiment,for the proof of concept.Here we found a research article, which use liner RNA with MS2 and PP7 aptamer, to do the split EGFP experiment.Below are their experimental data.
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<br>[[Image:The_principle_of_spite_EGFP.png  | border | center | 300px]]
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<br><center><font size="1">Figure 3.the principle and experimental data of split EGFP</font>
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We are SYSU-CHINA 2021.This year, we utilize circRNA as molecular scaffold to colalize enzymes, through the interaction of RNA aptamers and RNA binding proteins.For the proof of concept, we planed to perform split EGFP, which is an usual method to detect protein-protein interaction. If we can demonstrate that two EGFP fragments can be dragged to each other through the interaction between RNA aptamer and RNA binding proteins, which are linked to the fragments, then we can say that our concept can be proved. The mechanism lists below:
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<br>|image [[Image:Mechanism of split EGFP.png | border | center | 400px]]
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<center><font size="1">Figure 1.mechanism of split EGFP</font></center>
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EGFP splits into to part: EGFP-N and EGFP-C. EGFP-N is fused to MS2 while EGFP-C is fused to PP7, respectively. There is MS2 aptamer and PP7 aptamer on our circRNA scaffold, which have a 10nt length spacer. After the binding of two RBPs, their fused EGFP fragments can be dragged closer and form a complete EGFP. Then we can perform FCM(flow cytometry) to detect brightness.
 +
Our experiment included four groups, which are positive control(only transfected with plasmid which can express EGFP), negative control(transfected with nothing), experimental group(transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7 and circRNA scaffold 2), false positive group(transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7 but no circRNA), respectively. The results are as follows:
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|image [[Image:FCS1.png | border | center | 400px]]
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<center><font size="1">Figure 2.positive control</font></center>
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|image [[Image:FCS_Negative_control.png | border | center | 400px]]
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<center><font size="1">Figure 3.negative control</font></center>
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|image [[Image:FCS3.png | border | center | 400px]]
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<center><font size="1">Figure 3.co-transfection with circRNA scaffold</font></center>
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|image [[Image:FCS4.png | border | center | 400px]]
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<center><font size="1">Figure 4.co-transfection without circRNA scaffold</font></center>
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Result shows that the brightness of group4 is between group1 and group2, which conforms to our design. Brightness of group3 is as low as group1, indicating that no false positive effect are interfering our result. All in all, this split EGFP proves that our circRNA scaffold do work.
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Latest revision as of 15:10, 19 October 2021

loading sequence of RNA phage MS2

This sequence is necessary for loading of RNA into the capsid of RNA phage MS2. The coat proteins of phage MS2 is known to aggregate around this sequence.

Please see [http://2010.igem.org/Team:UT-Tokyo/Sudoku_assay_MS2 Phage MS2] assay page.


We are SYSU-CHINA 2021 and this year we use circRNA to colocalize enzymes, which are fused to RNA binding proteins(RBP). We choose MS2 coat protein and PP7 coat protein as our RBP,and their aptamer on circRNA.For contribution, we predict its 2D and 3D structure,to present a visual impression.
Group: SYSU-CHINA 2021
Author: Zhao bingnan
Summary: the 2D and 3D sturcture and application of MS2 aptamer
resource:2018_Book_VirusProteinAndNucleoproteinCo,Organization of Intracellular Reactions with Rationally Designed RNA
Assemblies
Uploads: MS2 aptamer on the circRNA scaffold.png;SYSU-CHINA circRNA-1.png;SYSU-CHINA circRNA-2.png;The principle of spite EGFP.png;MS2 binding mechanism.png

Applications of BBa_K313010

We are SYSU-CHINA 2021 and this year we use circRNA to colocalize enzymes, which are fused to RNA binding proteins(RBP). We choose MS2 coat protein and PP7 coat protein as our RBP,and their aptamer on circRNA.For contribution, we predict its 2D and 3D structure,to present a visual impression.

2D stucture

UNIQ33fb577f56e99b01-partinfo-00000001-QINU UNIQ33fb577f56e99b01-partinfo-00000002-QINU
We use RNAfold and StructureEditor to predict 2D structure of our circRNA, which contains MS2 aptamer.The results are below:

MS2 aptamer on the circRNA scaffold.png
Figure 1.2D stucture predicted by RNAfold

SYSU-CHINA circRNA-1.png
Figure 2.2D stucture predicted by StructureEditor

3D stucture

UNIQ33fb577f56e99b01-partinfo-00000003-QINU UNIQ33fb577f56e99b01-partinfo-00000004-QINU
We use this website:http://biophy.hust.edu.cn/new/3dRNA/create, to predict the 3D structure of our circRNA, which contains MS2 aptamer.The result lists below:


SYSU-CHINA circRNA-2.png

Figure 3. 3D stucture of MS2 aptamer predicted by Xiaolab

MS2 binding mechanism

UNIQ33fb577f56e99b01-partinfo-00000005-QINU UNIQ33fb577f56e99b01-partinfo-00000006-QINU


MS2 binding mechanism.png

Figure 4. MS2 binding mechanism


split EGFP

UNIQ33fb577f56e99b01-partinfo-00000007-QINU UNIQ33fb577f56e99b01-partinfo-00000008-QINU
We are SYSU-CHINA,and this year we use this part as MS2 coat protein's aptamer to do a split EGFP experiment,for the proof of concept.Here we found a research article, which use liner RNA with MS2 and PP7 aptamer, to do the split EGFP experiment.Below are their experimental data.


The principle of spite EGFP.png

Figure 3.the principle and experimental data of split EGFP

We are SYSU-CHINA 2021.This year, we utilize circRNA as molecular scaffold to colalize enzymes, through the interaction of RNA aptamers and RNA binding proteins.For the proof of concept, we planed to perform split EGFP, which is an usual method to detect protein-protein interaction. If we can demonstrate that two EGFP fragments can be dragged to each other through the interaction between RNA aptamer and RNA binding proteins, which are linked to the fragments, then we can say that our concept can be proved. The mechanism lists below:


|image
Mechanism of split EGFP.png
<center>Figure 1.mechanism of split EGFP

EGFP splits into to part: EGFP-N and EGFP-C. EGFP-N is fused to MS2 while EGFP-C is fused to PP7, respectively. There is MS2 aptamer and PP7 aptamer on our circRNA scaffold, which have a 10nt length spacer. After the binding of two RBPs, their fused EGFP fragments can be dragged closer and form a complete EGFP. Then we can perform FCM(flow cytometry) to detect brightness. Our experiment included four groups, which are positive control(only transfected with plasmid which can express EGFP), negative control(transfected with nothing), experimental group(transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7 and circRNA scaffold 2), false positive group(transfected with plasmids that can express EGFP-N-MS2 and EGFP-C-PP7 but no circRNA), respectively. The results are as follows:

|image
FCS1.png
Figure 2.positive control
|image
FCS Negative control.png
Figure 3.negative control
|image
FCS3.png
Figure 3.co-transfection with circRNA scaffold
|image
FCS4.png
Figure 4.co-transfection without circRNA scaffold

Result shows that the brightness of group4 is between group1 and group2, which conforms to our design. Brightness of group3 is as low as group1, indicating that no false positive effect are interfering our result. All in all, this split EGFP proves that our circRNA scaffold do work.

UNIQ33fb577f56e99b01-partinfo-00000009-QINU