Difference between revisions of "Part:BBa K342003:Design"

Revision as of 20:23, 5 November 2010

OmpR234 protein, with higher effect on Curli promoter

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, in order to ship the new part to iGEM HQ.

Source

PCR on genomic DNA from PHL818 strain.

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.

Forward primer sequence: 5'-TTGAATTCGCGGCCGCTTCTAGAAGGAGGTATATAATGCAAGAGAACTACAAGATTC-3'
Reverse primer sequence: 5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATCATGCTTTAGAGCCGTCCG-3'

A PCR product about 790 pb was expected and the good band had been extracted on agarose gel with a kit. The final part sent to the registry is a construction of the ompR234 BioBrick, cloned into pSB1C3.

References