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Team:Groningen/Promoters draft
From 2010.igem.org
Promoters
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth.
srfA
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html srfA operon] has been reported to be important for natural competence and sporulation in Bacillus Subtilis. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the ComXPA quorum sensing system and hence active in states of high cell densities. Therefore the srfA promoter would be suitable for chaplin expression. Two different lengths of the srfA promoter where chosen due to uncertainties concerning the region between the response element ant the transcription start side of the srfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one.
yqxM
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html yqxM-sipW-tasA] operon is controlled by the yqxM promoter. It is needed for biofilm formation because tasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including srfA, in response to quorum sensing. Since the caplins should work in a similar way to tasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 yqxM] promoter would be very suitable for chaplin expression during the stationary phase.
Quorum sensing and promoter interactions
References
Stöver AG, Driks A. Regulation of synthesis of the Bacillus subtilis transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ J Bacteriol. 1991 Sep;173(17):5487-93.]
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in Bacillus subtilis [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ Mol Microbiol. 2008 June; 68(5): 1117–1127.]
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in Bacillus subtilis. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/?tool=pubmed J Bacteriol. 2005 Oct;187(19):6659-67.]
