Part:BBa_K3960011

EGFP(N)-MS2

a chemeric gene whose N terminal is EGFP(N) and C terminal is MS2

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 475
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 823
  

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

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

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.

expression in HEK293

We separately transfect the plasmid which contain EGFP-N-MS2 and EGFP-C-PP7, respectively, into HEK293, to test whether our split EGFP fragment have expressed or not, to prevent the interference of false negative result. Western blot shows that our destinate fusion protein have both expressed in HEK293.

|image

Figure 5.western blot to detect expression of two EGFP fragment-RBP fusion protein in HEK293

The left lane represents EGFP-N-MS2 while the right lane represents EGFP-C-PP7.Result shows that the two fusion proteins have successfully expressed in HEK293.