Coding

Part:BBa_K2675003

Designed by: Esteban Lebrun   Group: iGEM18_Evry_Paris-Saclay   (2018-10-05)
Revision as of 15:46, 11 October 2018 by ELebrun (Talk | contribs)


PelB-SAIRGA

This part is the mature arbitrium peptide of phage phi3T (SAIRGA) preceded by the PelB secretion signal.

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 lysogenic-to-lytic viral cycle. By acting as a negative regulator of AimR, the SAIRGA signal makes the lysogeny-to-lysis switch of phi3T phage dependent on the “quorum” of 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 PelB secretion signal know to function in E. coli and thus designed this part.

To express this PelB-SAIRGA in E. coli, the sequence was codon optimized for E. coli DH5α, a specific RBSs (BBa_K2675013) 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_K2675043 was generated. This SAIRGA expression part did not behaved as predicted: it was not able to produce and release the mature hexapeptide SAIRGA in the culturing media of E. coli cells (for further details, visit the BBa_K2675043 page in the registry).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 54
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None