Introduction

This year the iGEM Team Düsseldorf worked on achieving the gold medal on the improvement of the L-rhamnose-inducible promoter pRha which is registered in the iGEM Registry as BBa_K914003. Part:BBa K9140031 The original part is an operon with the rhamnose inducible promoter pRha which regulates the expression of a fluorescence protein (in our case it will be mVenus) depending on the available L-rhamnose concentration. This system was reported for Synechocystis sp. PCC 6803 by Kelly et al. in 20182.

The improvement

Our approach to improving the construct refers to an approach that was already documented by Behle et al. in 20193 using Synechocystis. In addition to the promoter pRha and the fluorescence gene, our construct has the additional activator rhaS, which should lead to a sharp increase in the expression of the protein. With our approach, we want to ultimately prove with measured data that the activator rhaS has a positive influence on protein expression. In addition, the activator rhaS has so far only been occured in the genome of E. coli. As a result, the potential of this construct has so far been limited exclusively to this organism. By integrating the activator on the plasmid DNA, however, many new application possibilities are achieved, since the entire regulatory system could now also be used into other microorganisms.

Figure 1: The image shows the original construct pSHDY_prha-mVenus which contains the inducible promoter *pRha*, the fluorescent gene mVenus and a resistance gene for the antibiotic spectinomycin.

Figure 2: The image shows the improved construct pSHDY_Prha-mVenus_119rhaS that has already integrated the activator *rhaS*. It also contains the promoter *pRha* and codes for the fluorescent protein mVenus and a resistance gene for the antibiotic spectinomycin.

Results

We cloned and transformed E. coli MACH1 cells with the generated constructs. In figure 2 you can see colonies which are transformed with the construct containing the promoter pRha and the activator rhaS.

Figure 3: First transformation of the plasmid in *E.coli* with the additional activator *rhaS*.

Figure 4: shows the plate with positive transformed *E.coli* colonies. This can be seen on the plate, which is illuminated by UV light. It can be clearly seen that only a few colonies carry the activator, because they show a strong fluorescent signal. Most colonies do not show this reaction because they have the activator only integrated in their genome and not on their plasmid DNA.

Figure 5: Both pictures show the same plate. The right picture also shows the colonies under the influence of UV light, whereby some colonies do not show a strong fluorescence signal because they do not contain the additional activator in their plasmid DNA and therefore also do not express the fluorescent protein mVenus as strongly as colonies that have the plasmid with the activator integrated. The only two colonies that show a fluorescent signal are circled in red in the right image. They still carry the activator on the plasmid DNA.

So far we have unfortunately not been able to carry out further tests with the different colonies. The next step would be to incubate two bacterial cultures, one will contain the original pRha without that additional activator on the introduced plasmid and the other one will contain both parts. The mVenus fluorescence can be measured in a microplate reader. For this purpose, the concentration of the inducer rhamnose can be varied as desired. The variation in the rhamnose concentration helps with a more precise analysis of the influence on the system to be induced. It is also important to use an empty vector control (EVC) as a standard for the measurements (see Behle at al.)3. The EVC is the same E.coli strain, transformed with the same vector which does not contain the activator rhaS and the fluorescence gene mVenus.

The expression rate of the mVenus can then be evaluated because of the mVenus response under the influence of UV-light. With the results obtained, we will be able to deduce whether the activator rhaS has a positive influence on the expression rate and whether it would thus improve the whole construct. An outstanding advantage of our improvement is that the activator rhaS is just found in E. coli, but not in other organisms' genomes. With the integration of the activator rhaS into the plasmid already containing the pRha promoter, this inducible system can now be used in many other microorganisms. This offers a universal application and a large variety of possibilities for cloning this construct into other bacteria that do not have the activator in their natural genome. However, the improved system is not only applicable to microorganisms. After transformation into agrobacteria, the construct can also be transferred to higher organisms such as plants.

Assumptions

Our expectation is that the protein mVenus has a much higher expression rate with the help of the integrated activator rhaS in the plasmid and thus shows a much stronger fluorescence signal. The cells with the plasmid without the activator rhaS will send a weaker signal, but the activator in the genome will cause the mVenus gene to be further expressed. The EVC standard should not show a fluorescent signal, since these cells do not have the mVenus gene in their plasmid. We take this assumption from experiments carried out by Behle et al.3 which, however, were done in Synechocystis.

References

1. L-rhamnose-inducible promoter (pRha). Registry of Standard Biological Partst.

   (2012-09-20). Retrieved on October 21 , 2021. from parts.igem.org

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2. Kelly, C. L., Taylor, G. M., Hitchcock, A., Torres-M ndez, A., & Heap, J. T. (2018).

   A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria.

   ACS Synthetic Biology 7(4), 1056-1066.

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3. Behle, A., Saake, P., & Axmann, I. M. (2019).

   Comparative analysis of inducible promoters in cyanobacteria.

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