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Team:Uppsala/promoters
From 2013.igem.org
Constitutive promoter library for Lactobacillus & E.coli
Introducing new constitutive promoters to the iGEM community
Depending on how much you want to express your gene, you will need promoters of different strengths. Therefore we wanted to provide a selection of promoters that work in both E. coli and Lactobacillus. We synthesized seven promoters based on the CP promoter library in the article[1]. These promoters are based on consensus sequences from promoters in Lactococcus lactis. Below you can see our promoters and their name in the part registry.
Strength measuring of CP1 and CP41 in E. coli using FACS. – The peaks show the median of how much the
cells in the sample fluoresced.
To characterize them we assembled them with the reporter gene BBa_K592024 B0034-BFP in pSB3K3 and transformed them into E. coli. BFP stands for “Blue Fluorescence Protein” and the expression of this gene leads to production of a fluorescence protein which we can measure with a fluorescence-activated cell sorting (FACS) machine. The FACS gives you a value of the fluorescence from each cell in your sample. As a reference we used the promoter J23101 from the standard parts in the registry, where its strength were put to 1.
The CP promoters strength relative to J23101 in E. coli.
From this we got the relative strength of our promoters, compared to J23101. The weakest promoters in our collection have half the strength of J23101, while the strongest promoter is approximately four times stronger.
Further characterization of CP promoters in Lactobacillus
Because of time constraints, we have not had the time to characterize them in Lactobacillus. Though in the future we plan to do this by first sub cloning them together with BBa_K592024 B0034-BFP from our previous assemblies and then assemble them with our shuttle vector. After that we can transform them into either Lactobacillus reuteri or Lactobacillus plantarum and characterize them in the same way as we did in E. coli. The only difference will be that we won’t have any reference, since J23101 doesn’t work in L. reuteri or L. plantarum. We will only get the strength of each promoter relative the strength of our other CP promoters.In the article by Jensen and Hammer they stated that these promoters were based on the consensus sequence of promoters in gram-positive bacterium L. lactis, though there are no obvious reasons why they shouldn’t work in other organisms. Jensen and Hammer characterized them in both L. lactis and gram-negative bacterium E. coli using the reporter gene lacLM. This indicates that there is a possibility that these promoters are near universally applicable to prokaryotic organisms in general.
References
[1]. The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters PETER RUHDAL JENSEN AND KARIN HAMMER Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark 21 October 1997 - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/pdf/am000082.pdf
