Part:BBa_K2675002
OmpA-SAIRGA
This part is the mature arbitrium peptide of phage phi3T (SAIRGA) preceded by the OmpA secretion signal.
It is an improvement of BBa_K2279001.
Usage and Biology
The hexapeptide SAIRGA is the signalling molecule used for cell-to-cell communication [1]. To perform its quorum sensing function in the natural phi3T phage infection, SAIRGA needs to be secreted out of the cell. It is produced as an immature pre-pro-peptide, AimP (BBa_K2279001), that upon secretion is cleaved extracellularly to remove the secretion signal and release the mature hexapeptide. The mature peptide then enters cells and binds to its receptor protein AimR (BBa_K2279000 and BBa_K2675000). Binding of SAIRGA to AimR blocks the activator function of AimR that, in turn, facilitates a switch from lytic-to-lysogenic viral cycle. By acting as a negative regulator of AimR, the SAIRGA signal makes the lysis-to-lysogeny switch of phi3T phage dependent on the “quorum” of phi3T phages in the bacterial populations.
In phi3T phage, SAIRGA is produced with specific secretion and protease-cleavage tags [1] that allow secretion and extracellular processing by Bacillus. Since we had no further information about this protease, we could not investigate the ability of Escherichia coli to produce a similar enzyme. Consequently, to produce SAIRGA in E. coli, we have decided to replace this tag by the OmpA secretion signal know to function in E. coli and thus designed this part.
To express this OmpA-SAIRGA in E. coli, the sequence was codon optimized for E. coli DH5α, a specific RBSs (BBa_K2675012) was designed with the Salis RBS Calculator [2, 3] and the peptide was placed under the control of the constitutive strong promoter (BBa_J23100). Thus, the composite part BBa_K2675042 was generated. This SAIRGA expression part behaved as predicted: it was able to produce and release the mature hexapeptide SAIRGA in the culturing media of E. coli cells (for further details, visit the BBa_K2675042 page in the registry).
References
[1] Erez Z, Steinberger-Levy I, Shamir M, Doron S, Stokar-Avihail A, Peleg Y, Melamed S, Leavitt A, Savidor A, Albeck S, Amitai G, Sorek R. Communication between viruses guides lysis-lysogeny decisions. Nature (2017) 541, 488-493.
[2] Espah Borujeni A, Channarasappa AS, Salis HM. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res (2014) 42, 2646-2659.
[3] Salis HM, Mirsky EA, Voigt CA. Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol (2009) 27, 946-50.
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]
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