Part:BBa_K2675044

Tat-SAIRGA expression cassette

This part is the mature arbitrium peptide of phage phi3T (SAIRGA) preceded by the Tat secretion signal (BBa_K2675004), equipped by a synthetic RBS (BBa_K2675014) and placed under the control of the constitutive strong promoter BBa_J23100 and of the strong terminator L3S2P21 (BBa_K2675031).

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 and BBa_K2675001), 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 phage in Bacillus cells.

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 Tat secretion signal of the csp2 gene of Corynebacterium glutamicum (Uniprot Q04985) know to function in E. coli.

To express the Tat-SAIRGA in E. coli, the sequence was codon optimized for E. coli DH5α, a specific RBS (BBa_K2675014) was designed with the Salis Lab RBS Calculator [2, 3] and the peptide was placed under the control of the constitutive strong promoter (BBa_J23100) and of the strong terminator L3S2P21 (BBa_K2675031). Thus, this composite part (BBa_K2675044) was generated.

This SAIRGA expression part did not behave as predicted: it was not able to produce and release detectable levels the mature hexapeptide SAIRGA in the culturing media of E. coli cells.

For more information, please visit http://2018.igem.org/Team:Evry_Paris-Saclay/Peptide.

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

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]