Coding

Part:BBa_K1349001:Design

Designed by: Alexia Satouf, Clara Bouyx, Aimeric Agaoua, Lambert Antoni, Vincent Castel   Group: iGEM14_Aix-Marseille   (2014-10-06)

relA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1708


Design Notes

This part is designed to allow the rapid synthesis of the alarmone ppGpp, responsible for the stringent response in E. coli. In the context of our project ppGpp is accumulation used to stop initiation of cell division. This part is complementary to part BBa_K1349004.

Expression is expected to cause accumulation of ppGpp and so reduce growth rate. Expression in a mutant unable to make ppGpp is expected to complement the mutant. This complementation has been tested and is observed.

Our RelA brick (BBa_K1349001) represents a significant improvement on the previously available RelA brick made by the 2011 Trondheim team (Part:BBa_K639001). In the first place our brick has no amino acid mutations and no upstream unwanted insert. Secondly our brick lasks the PstI site and thus is compatible with the assembly standards RFC10, 12, 21, 23, 25 and 1000. We have shown that out brick is active when expressed in E. coli and able to complement a relA mutant.

Source

The part was obtained by PCR from the pT18 RelA provided by Emmanuelle Bouveret and SLIC assembly. The construction removed the PstI restriction site in the gene by the silent mutation of a CTG codon to a CTC codon. The construction contains the entire coding sequence.

References