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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.



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 cant’t be sure if J23101 will work in L. reuteri or L. plantarum. In that case we will only get the strength of each promoter relative the strength of our other CP promoters.

These promoters have further been used successfully in several parts in our project, including BBa_K1033000 (TAL), BBa_K1033002 (STS) and BBa_K1033281 (amilCP).

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