A constitutive promoter (CP) library for Escherichia coli and Lactobacillus genus

Introducing new constitutive promoters to the iGEM registry

Depending on how much you want to express your gene, you will need promoters with different strengths. Therefor we wanted to provide a selection of promoters that works in both E. coli and Lactobacillus genus. We synthesized seven promoters based on the CP promoter library in the article “The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters." by Peter Ruhdal Jensen and Karin Hammer. These promoters are based on the consensus sequence in promoters from Lactococcus lactis. Bellow you can see our promoters and their name in the part registry.

CP1 - BBa_K1033219

CP8 - BBa_K1033220

CP11 - BBa_K1033221

CP29 - BBa_K1033222

CP30 - BBa_K1033223

CP41 - BBa_K1033224

CP44 - BBa_K1033225

Strength measuring of CP1 and CP41 in E. coli using FACS. – The peaks show the median of how much the cells in the sample fluorescence.

To characterize them we assembled them with the reporter gene B0034-BFP in pSB3K3 and transformed them into E. coli. BFP stands for “Blue Fluorescence Protein” and the expression of this gene lead 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

Because of time constrains, we have not had the time to characterize them in any Lactobacillus. Thought in the future we plan to do this by first subclone them together with 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 originally taken from 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 universally applicable to prokaryotic organisms in general.