Project/SpooA/

From 2010.igem.org

Revision as of 17:33, 24 October 2010 by Greg M (Talk | contribs)


Contents

SpooA repressor - [http://partsregistry.org/Part:BBa_K354004 BBa_K354004]

BioBrick Background

So as to inhibit the transcription driven by the PlcR promoter, the SpoOA protein binds to a particular sequence. Binding to this region prevents polymerase from properly interacting with the promoter and thus represses transcription. The regions within the PlcR promoter that SpoOA binds are referred to as the SpoOA boxes, of which, two of these boxes flank the PlcR-PapR positive regulator binding site thus repressing transcription [1].

Sequence Development and Annotation

The SpoOA repressor was synthesized and and delivered within the pMK plasmid. So as to use SpoOA in development, it was excised from the plasmid via digestion with EcoR1 and Pst1.
PMK-SpoOA -real.JPG

Once digested, the SpoOA fragment was gel extracted and purified in preparation for ligation into PSB1C3. The SpoOA insert (442bp) can be clearly seen. <bp /> SpoOA digest.JPG

The retrieved SpoOA fragments were ligated into the PSB1C3 linearised plasmid and then re-digested with EcoR1 and Pst1 so as to confirm the insertion.
SpoOA confirmation digest.JPG

The SpoOA-PSB1C3 plasmid map is shown below as well as all necessary restriction sites and landmark annotations [2].
PMK-SpoOA.JPG

Testing and verification


References

[1] Lereclus D., Agaisse H., Grandvalet C., Salamitou S. & Gominet M. (2000), Regulation of toxin and virulence gene transcription in Bacillus thuringiensis. Int. J. Med. Microbiol. 290:295-299.