Part:BBa_K5090004
Description
P2A
P2A is a part of the 2A family of peptides found in the foot-and-mouth-disease virus. 2A peptides are usually 19-22 amino acids located between the sequences of two proteins. Known as a “self-cleaving peptide”, 2A proteins induce ribosome skipping, usually between the last glycine-proline sequence at the C-terminal of the 2A peptide (Wang et al., 2015). According to their paper, the peptide bond is not formed between that glycine and proline and the ribosome continues translation at the next amino acid. This leaves behind a 1-amino acid scar on the protein C-terminal to the 2A peptide and an 18-21 amino acid scar on the protein N-terminal to the 2A peptide. This essentially generates two proteins from a single promoter and mRNA strand. 2A peptides with a GSG (Gly-Ser-Gly) sequence in advance of the 2A sequence, as this part does, have been found to have a higher cleavage efficiency (Wang et al., 2015).
This part was derived from Molinari (2019).
Usage and Biology
P2A allows for the generation of two separate, independent proteins under a single promoter. This is beneficial as it allows for the expression of both proteins to be controlled together. The 2024 Stony Brook iGEM used P2A in between the sequences of the transcriptional and translational repressor proteins LacI (BBa_K5090001) and L7Ae (BBa_K5090002). This lets LacI and L7Ae be expressed under the same promoter. As our gene circuit involved adding a miRNA target site to our repressor proteins, only a single miRNA target site could suppress both LacI and L7Ae with P2A. This helps ensure the suppression of both repressors by microRNA at the same time. This is based on the design of Wang et al. (2023). Wang et al. (2023) characterized their dual regulation system in mammalian cells, using P2A in between the sequence of LacI and L7Ae.
In one of our several designs of the dual regulation system, we elected to add the P2A sequence after the miRNA target site that is in the coding sequence of LacI (BBa_K5090007). As bacterial systems lack the post-transcriptional modifications that are present in eukaryotic cells, simply removing a part of the 5’ or 3’ UTR is not enough to inhibit translation. Thus, we elected to put the miRNA target site at several different sites, one of which included after the start codon of LacI, thus in its coding sequence. Cleavage at this site would remove the RBS, thereby inhibiting translation. Including the target site after the start codon would affect the coding sequence of LacI, adding in the P2A sequence after the target site would work to minimize the effects on LacI’s coding sequence. Instead of a 7-amino acid residue from the target site, there would only be a 1-amino acid scar on LacI, as P2A leaves behind a single proline on the N-terminal of the peptide following ribosome skipping.
Characterization
Our plasmid containing LacI and L7Ae containing both P2A sites and the miRNA target site was successfully cloned using TEDA without any errors or mutations in the coding sequence. As described above, one P2A sequence is located between the target site and the coding sequence of LacI, and the second P2A sequence is located between the coding sequences of LacI and L7Ae.
Figure 1: Plasmid showing successful cloning of P2A.
Further, we can confirm that the LacI from which this P2A separated was functional, as there is a statistically significant difference between GFP (BBa_K5090003)
expression in the case where GFP expressed alone and the case where GFP was expressed alongside its repressors, per the description above.
Figure 2: Graphs showing successful expression of LacI and L7Ae in decreasing fluorescence when LacI is preceded by P2A and a P2A is placed between LacI and L7Ae coding sequences.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- Wang, Y., Wang, F., Wang, R., Zhao, P., & Xia, Q. (2015). 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci Rep, 5, 16273. https://doi.org/10.1038/srep16273
- Shu, W.-J., Lee, K., Ma, Z., Tian, X., Jong Seung Kim, & Wang, F. (2023). A dual-regulation inducible switch system for microRNA detection and cell type-specific gene activation. Theranostics, 13(8), 2552–2561. https://doi.org/10.7150/thno.84111
- Molinari, S., Shis, D. L., Bhakta, S. P., Chappell, J., Igoshin, O. A., & Bennett, M. R. (2019). A synthetic system for asymmetric cell division in Escherichia coli. Nature Chemical Biology, 15(9), 917–924. https://doi.org/10.1038/s41589-019-0339-x
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