Introduction

Inducible systems able to sense and respond to fluctuation of a compound of interest, are some of the most practical tools synthetic biology has to offer. However, such systems are not easy to find and, more often than not, are inadequately characterized. During this year’s competition, our team tried to characterize a major component of such a system, the FR1 promoter, the actuator of the malonyl-CoA sensor-actuator system we have implemented in our design ^[1].

Biology

P_FR1 is a synthetic promoter containing binding sites for the malonyl-CoA-responsive transcription factor FapR (BBa_K4624003) (Fig. 1), derived from the Gram-positive bacteria Bacillus subtilis ^[2]. In the presence of low malonyl-CoA levels, FapR binds to FapRO sites and inhibits the promoter. While, the binding of malonyl-CoA to FapR triggers a conformational change to the protein, causing FapR-DNA complex dissociation ^[3].

Figure 1: Sequence of the engineered malonyl-CoA-responsive FR1 promoter; Bold sequences represent the -35 and -10 regions; FapR-binding sites are underlined. Table was adapted from [1].

Characterization plan

To adequately characterize the promoter we had to shed light on two aspects:

1. the strength of the synthetic P_FR1 , compared to a standard one, such as an Anderson promoter and

2. the expression levels of FapR needed to effectively regulate the promoter.

Therefore, we planned to design, build and test 2 constructs for an initial characterization of the system:

For the 1st experiment, a level 1 (alpha) construct with the P_FR1 upstream the reporter syfp2 was designed for promoter strength evaluation. By comparing it to a standard promoter, we aimed to provide future users with a straightforward way to assess the suitability of this promoter for their specific purposes.

For the 2nd experiment, a level 2 (omega) construct was designed, containing the aforementioned level 1 (alpha) reporter construct and an additional one, carrying the fapR regulated by the AraC/P_BAD system. So, by titrating arabinose to control the levels of FapR we will be able to determine the levels of repression the protein exerts on the promoter, and the degree of fapR transcription required to make the system responsive.

Experimental Design and Results

To begin, we domesticated the sequences of the FR1 promoter, the AraC/P_BAD system, and fapR. The domestication involved removing internal restriction sites that were not compatible with the GoldenBraid cloning method and the addition of appropriate 3' and 5' 4-nt overhangs. Once designed, the domesticated sequences were ordered to be synthesized by IDT.

The next step was to proceed with the assembly. The domesticated sequences were inserted into a pUPD2 part domestication vector to create level 0 constructs that we could then combine to assemble our complete constructs. Once the insertion was verified through restriction-digestion reaction and gel electrophoresis (Fig. 4), we used the resulting level 0 constructs along with the ones of the B0030 RBS (BBa_J428032), syfp2 (BBa_K864100) and B0015 double terminator (BBa_J428092), provided by this year’s iGEM distribution kit, to create the level 1 reporter construct (Fig. 2) and the level 1 construct carrying fapR. Both constructs were successfully assembled, and the inserts were once again confirmed through restriction-digestion reactions (Fig. 5). Subsequently, we utilized the reporter construct for the P_FR1 in the 1st experiment.

1st experiment

Having the level 1 (alpha) reporter construct (Fig. 2) ready, we set out to perform the 1st experiment. In short, the isolated plasmid carrying the reporter construct was used to transform E. coli BL21 (DE3) chemically competent cells, following standard protocol. Single colonies were picked to inoculate 5 ml of LB medium, with the appropriate antibiotic, and cultured O/N at 37^οC and 210 rpm. Single colonies were also picked for the positive control (syfp2 under the regulation of the Anderson J23118 promoter), and the negative control (non-transformed E. coli BL21 DE3 cells). The O/N cultures were used to prepare the final dilutions into M9 minimal medium in order to reach the same starting OD₆₀₀. Measurements were taken after 6 hours of incubation (37^οC and 210 rpm) at 511 nm (excitation) and 529 nm (emission) (Fig. 6).

It is evident that the P_FR1 exhibits a fluorescence output similar to that of the constitutive P_J23118, in absence of the FapR regulatory protein. So we can conclude that these two promoters have a comparable intensity.

2nd experiment

Now that we had tested the functionality of the level 1 (alpha) reporter construct and provided an estimate for the promoter's strength we could start the process of building the level 2 (omega) construct to assess the relation of P_FR1 with its regulatory protein FapR. To achieve that, we combined the construct from the 1st experiment, with the level 1 (alpha) construct carrying the fapR under the control of the AraC/P_BAD system. Using this construct, we can employ various arabinose concentrations to induce varying expression levels of fapR and observe how P_FR1 responds to this regulation. Gathering this type of information is essential for integrating both components into any regulatory circuit, similar to what we have integrated in oPHAelia’s design.

The construct was assembled following our standard protocol for digestion-ligation reaction. After a few failed attempts, we managed to successfully build the level 2 construct which was again confirmed with restriction-digestion reaction and gel electrophoresis (Fig. 7).

Similarly to the 1st experiment, we started again by transforming E. coli BL21 (DE3) chemically competent cells with the isolated plasmid carrying the level 2 construct. The next day, for the preparation of liquid cultures, single colonies were picked and inoculated in LB medium, with the appropriate antibiotic, and finally the cultures were incubated O/N at 37^οC and 210 rpm. The next morning, final dilutions x5 were prepared in M9 minimal medium for the level 2 construct as well as for the same positive and negative controls used in the 1st experiment. Lastly, addition of L-arabinose followed for the preparation of 4 different final concentrations (0.01, 0.1, 1 and 10 mM), creating various levels of P_BAD induction. Measurements at wavelengths of 511 nm (excitation) and 529 nm (emission) were taken at 3h and 6h timepoints (Fig. 8).

As we can see from the results (Fig. 8), there is a significant decrease of fluorescence intensity at increasing concentrations of L-arabinose. This fact confirms the existence of the FapR-mediated P_FR1 repression, since higher concentration of L-arabinose implies a higher activation of the P_BAD, through the AraC regulatory protein, and ultimately a higher expression of fapR.

References

1. Liu D, Xiao Y, Evans BS, Zhang F. Negative feedback regulation of fatty acid production based on a malonyl-CoA sensor-actuator. ACS Synth Biol. 2015 Feb 20;4(2):132-40. doi: 10.1021/sb400158w. Epub 2014 Jan 10. PMID: 24377365.

2. Schujman GE, Paoletti L, Grossman AD, de Mendoza D. FapR, a bacterial transcription factor involved in global regulation of membrane lipid biosynthesis. Dev Cell. 2003 May;4(5):663-72. doi: 10.1016/s1534-5807(03)00123-0. PMID: 12737802.

3. Schujman GE, Guerin M, Buschiazzo A, Schaeffer F, Llarrull LI, Reh G, Vila AJ, Alzari PM, de Mendoza D. Structural basis of lipid biosynthesis regulation in Gram-positive bacteria. EMBO J. 2006 Sep 6;25(17):4074-83. doi: 10.1038/sj.emboj.7601284. Epub 2006 Aug 24. PMID: 16932747; PMCID: PMC1560364.