Difference between revisions of "Part:BBa K5131008"

Revision as of 19:47, 1 October 2024

FliPGFP_nsp5 inducible

Information of SARS-Cov-2 nsp5 can be seen in BBa_K5131003 or BBa_K5131003. This part together with BBa_K5131007 can be used as an in vivo inhibitor screening platform for nsp5. It can be activated by nsp5 to emit fluorescence.Due to the flexibility of FlipGFP, this part allows for detection of different proteases by simply replacing the protease recognition sequence. We believe this part offers an efficient and accurate reporter for any iGEM team aiming to detect protease activity.

Construction design

Previously, Wageningen_UR attempted to construct a FlipGFP recognized by MMP-9 in 2022. They successfully created and submitted the part (BBa_K4244045), but unfortunately, their FlipGFP was not activated by MMP-9 and did not emit fluorescence. To avoid the same situation, we carefully examined the sequence of the part they submitted and found that FlipGFP is located within only one ORF, and there are subtle differences between the sequence information and those reported in the literature. To obtain a FlipGFP that can be correctly activated, we used the sequence reported in the original literature[1], replacing the TEV protease recognition sequence with the nsp5 recognition sequence(N-AVLQSGFRK-C). To ensure the correct expression of FlipGFP, we used the pRSF-Duet1 and separately inserted the first nine β-strands of FlipGFP and the engineered 10-11 β-strands into two different ORFs(Figure 1A). This vector is referred to as pRSF-FlipGFP_nsp5(10-11)-FlipGFP(1-9). We constructed this vector through homologous recombination. The sequencing results confirmed the correct construction of our vector(Figure 2B).

Figure 1. A) Vector design of pRSF-FlipGFP_nsp5(10-11)-FlipGFP(1-9). B) Sequencing validation of FlipGFP_nsp5(10-11)-FlipGFP(1-9).

Genetic circuit design

To facilitate inhibitor screening, we designed a genetic circuit with the following functionalities (Figure 2): when only FlipGFP is present, the entire system does not emit fluorescence; when both nsp5 and FlipGFP are present, nsp5 cleaves FlipGFP, resulting in fluorescence; and when nsp5 is inhibited, the cleavage efficiency of nsp5 on FlipGFP decreases, leading to reduced or absent fluorescence.

Figure 2. Genetic circuit of nsp5 activated FlipGFP.

Proof of concept

We separately transformed E.coli BL21 with FlipGFP alone and co-transformed BL21 with FlipGFP and nsp5, then plated them on LB plates containing IPTG at a final concentration of 0.2 mM. The results showed that colonies transformed with FlipGFP alone produced almost no fluorescence, while colonies co-transformed with FlipGFP and nsp5 emitted significant fluorescence. This demonstrates that we successfully constructed a FlipGFP system that can be activated by nsp5 to emit fluorescence(Figure 3).

Figure 3. Activation of FlipGFP by nsp5.

Reference:
1. Zhang Q, Schepis A, Huang H, Yang J, Ma W, Torra J, Zhang SQ, Yang L, Wu H, Nonell S, Dong Z, Kornberg TB, Coughlin SR, Shu X. Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals. J Am Chem Soc. 2019 Mar 20;141(11):4526-4530. doi: 10.1021/jacs.8b13042. Epub 2019 Mar 6. PMID: 30821975; PMCID: PMC6486793. Sequence and Features