Difference between revisions of "Part:BBa K3960002:Experience"

Latest revision as of 14:30, 19 October 2021

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Applications of BBa_K3960002

User Reviews

UNIQ849e964ad7ca4c3e-partinfo-00000000-QINU UNIQ849e964ad7ca4c3e-partinfo-00000001-QINU 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

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

400px

Figure 2.positive control |image

Figure 3.negative control |image

Figure 3.co-transfection with circRNA scaffold |image

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.

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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:
+<br>|image [[Image:Mechanism of split EGFP.png | border | center | 400px]]
+<center><font size="1">Figure 1.mechanism of split EGFP</font></center>
+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 [[Image:Positive contriol.png | border | center | 400px]]
+<center><font size="1">Figure 2.positive control</font></center>
+|image [[Image:FCS_Negative_control.png | border | center | 400px]]
+<center><font size="1">Figure 3.negative control</font></center>
+|image [[Image:FCS3.png | border | center | 400px]]
+<center><font size="1">Figure 3.co-transfection with circRNA scaffold</font></center>
+|image [[Image:FCS4.png | border | center | 400px]]
+<center><font size="1">Figure 4.co-transfection without circRNA scaffold</font></center>
+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.