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In this year’s project, we contributed a total of 7 basic parts and 4 composite parts to the iGEM community. Our main contributions are BBa_K5131008 and BBa_K5131002.
BBa_K5131008 is a FlipGFP that can be correctly activated by proteases to emit fluorescence. This part enables detection of different proteases by simply replacing the protease recognition sequence. We believe this tool offers an efficient and accurate reporter for any team aiming to detect protease activity.
BBa_K5131002 is a rationally designed nsp5 mutant (nsp5-T21I), which can recognize and efficiently cleave specific protein sequences. We believe this part can be used as a tool enzyme for removing recombinant tags during protein purification. Compared to purchasing commercial proteases, BBa_K5131002 offers a more economical and efficient solution for tag removal.
All the new parts we contributed are listed in the following table:
| Part id | Name | Part type | Description |
|---|---|---|---|
| BBa_K5131000 | SARA-CoV-2 nsp5 | Coding | This part is SARA-CoV-2 nsp5 .We express this part with pGEX-6P-1.Through structure prediction, we investigate the reason why the N-terminal GST tag could not be cleaved by HRV 3C protease and to assess its impact on the enzymatic activity of nsp5. |
| BBa_K5131001 | SARA-CoV-2 nsp5_native | Coding | This part is SARA-CoV-2 nsp5 with 4 additional residues (AVLQ) at its N-terminus. We express this part with pGEX-6P-1 to get nsp5 in its native state. We characterized its catalytic efficiency as kcat/Km=27,691 s⁻¹M⁻¹. |
| BBa_K5131002 | SARA-CoV-2 nsp5_T21I | Coding | This part is a nsp5 mutant, mutation of the 21st amino acid of nsp5 from T to I. The rationally designed nsp5 exhibits 26.6% higher enzymatic activity compared to the wild-type, making it a promising tool enzyme for removing tags during protein purification. We hope this part will help all iGEMers reduce the cost of protein purification while improving efficiency |
| BBa_K5131003 | FlipGFP(1-9) | Reportor | This part includes the first 9 β-strands of FlipGFP and is a generic component of FlipGFP. |
| BBa_K5131004 | FlipGFP_nsp5 inducible(10-11) | Reporter | This part includes the 10-11 β-strands of FlipGFP where the protease recognition sequence linked before β11 was replaced with the nsp5 recognition sequence(N-AVLQSGFRK-C). |
| BBa_K5131005 | GST tag with HRV 3C protease cleavage site | Tag | This part is GST tag with HRV 3C protease cleavage site.GST tag. The GST tag promotes soluble expression of nsp5, and the presence of HRV 3C protease cleavage site allows the HRV 3C protease to remove the GST tag |
| BBa_K5131010 | pGEX Terminator(predicted) | Terminator | This part is a terminator on pGEX-6P-1. We predicted the most likely terminator sequence using iTerm-PseKNC in the region downstream of pGEX-6P-1 multiple cloning site to upstream of the Amp promoter. |
| Part id | Name | Usage |
|---|---|---|
| BBa_K5131006 | pGEX-GST-nsp5-His | Tests for expressing nsp5. |
| BBa_K5131007 | pGEX-GST-nsp5_native-His | For expressing SARS-CoV-2 nsp5 with its native N- and C-termini. |
| BBa_K5131008 | FliPGFP_nsp5 inducible | For in vivo detection of nsp5 enzymatic activity, which can be cleaved by active nsp5 to produce fluorescence. When used in conjunction with BBa_K5131002, it serves as an in vivo screening platform for SARS-CoV-2 nsp5 inhibitors. |
| BBa_K5131009 | pGEX-GST-nsp5_T21I-His | For expressing SARS-CoV-2 nsp5-T21II. |