Part:BBa_K342003:Design
OmpR234 protein, with higher effect on Curli promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 137
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Escherichia coli bacteria into biofilms contain many adherence structures, as curli structures composed by CsgB and CsgA proteins. Laboratory E. coli K12 strain hasn’t naturally got this phenotype. However, we had a mutant K12 MG1655 E. coli strain on which adherence phenotype had been observed. Indeed, there was a mutation in the genome of the bacteria, particularly in the ompR regulatory gene, at the position 234. The corresponding OmpR234 protein strongly activates the csgD promoter, and CsgD protein activates CsgB et CsgA transcriptions. So, we intended to create an ompR234 coding sequence BioBrick™ by PCR on this bacteria genome. Chromosomic DNA PHL818 strain had been extracted with DNeasy Blood & Tissue (QIAGEN) kit. Primers couple had been designed with iGEM extension, containing iGEM restriction enzymes sites: a forward primer with a perfect RBS (Ribosome Binding Site) and a reverse primer with a double transcription terminator. A PCR product about 790 pb had been extracted on agarose gel with NucleoSpin Extract II (Macherey-Nagel) kit or silica balls kit. Then, we made a cloning into pSB1C3 backbone, that we sent to the registry.
Source
PCR on genomic DNA from PHL818 strain.
Forward primer sequence: 5'-TTGAATTCGCGGCCGCTTCTAGAAGGAGGTATATAATGCAAGAGAACTACAAGATTC-3'
Reverse primer sequence: 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGCTTTAGAGCCGTCCG-3'
References
"Complex Regulatory Network Controls Initial Adhesion and Biofilm Formation in Escherichia coli via Regulation of the csgD Gene",Claire Prigent-Combaret, Eva Brombacher, Olivier Vidal, Arnaud Ambert, Philippe Lejeune, Paolo Landini, and Corinne Dorel, J Bacteriol. 2001 December