Part:BBa_K1670005

EsaR repressor/activator

BBa_K1670005 BBa_K1670005 encodes for the regulatory protein EsaR of the EsaR/I quorum sensing system from Erwinia stewartii . Contrary to most other quorum sensing systems, EsaR works as a repressor rather than an activator[1,2]. It binds at its corresponding binding box between the -10 and the -35 region of its corresponding promoter and inhibits transcription. Binding of 3OC6-HSL to EsaR induced an allosteric change in the structure that prevents its DNA-binding ability and thus induces expression. If the binding box is positioned shortly upstream of the promoter, EsaR also works as an activator of the respective promoter as long as it can bind to the DNA. We use a D91G variant of the gene that shows higher sensitivity towards 3OC6-HSL [1].

Characterization

BBa_K1670005 (esaR) is characterized along with BBa_K1670001 (P_esaRC_cfp) in a fluorescence-based assay. BBa_K1670005 is cloned into J61002_J23100 (Fig.1) and is afterwards cotransformed together with pSB3C5_BBa_K1670001 (Fig.2) into E.coli BL21.

Overnight cultures are inoculated and diluted to an OD of 0.02 and spotted on microtiter plates. Synthesis of mRFP is induced with different concentrations of 3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) ranging from a final concentration of 0.01nM – 100 nM. Cultures are incubated at 37°C in a platereader (Biotek Synergy MX) and fluorescence (Excitation 439 nm, Emission 476 nm) and OD₆₀₀is measured every 15 minutes. 10 seconds before measuring the plate is shaked (orbital, medium). As a positive control E.coli BL21 pSB3C5_J04421 diluted to an OD₆₀₀of 0.02 and induced with a final concentration of 0.1 mM IPTG are used. As negative controls and to account for possible auto fluorescence of the cells wild type E.coli BL21 is used. Also sterile controls containing only LB-media without any cells are pipetted on the microtiter plates.

Figure 1 - J61002_BBa_J23100_BBa_ K1670005 (esaR).

Figure 2 . -pSB3C5_BBa_K1670001 (P_esaRC_cfp)

Results

The constitutively expressed EsaR is expected to act as a repressor on P_esaRC. In the presence of 3OC6-HSL it should dissociate from the DNA and allow expression of the P_esaRC controlled CFP.

As seen in figure 3 the sample with 0.01 nM 3OC6-HSL had the highest emission at 476 nm of nearly 7000 RFU, followed by the 0 nM sample at 5000 RFU, the positive control at 4500 RFU sample, induced with 0.1 nM 3OC6-HSL, at around 3000 RFU. The other samples showed a very low emission at or under 1500 RFU.

Figure 3 Results of the CFP emission measurement (476 nm) of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670005 pSB3C5_BBa_K1670001 induced with 3OC6-HSL concentrations from 0.01 nM to 100 nM, 0 nM and the positive control for CFP.

For a better overview we compare 0.01 nM, 10 nM, 0 nM and the positive control more closely (Fig. 4). Here we can observe that the fluorescence of the 0.01 nM sample is the highest. The sample without any 3OC6-HSL and the positive control are at about the same level and the sample with 10 nM showed no increase in fluorescence at all. Those results suggest that EsaR ceases its repressor function at very low 3OC6-HSL concentrations of 0.01 nM but inhibit the expression at 3OC6-HSL concentrations higher than 10 nM. The relatively high level of basal CFP expression in the 0 nM sample suggests that P_esaR is not a very tightly regulated promoter.

Figure 4 Results of fluorescent emission measurement at 476 nm of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670005 pSB3C5_BBa_K1670001 induced with 3OC6-HSL at concentrations of 0.01 nM, 10 nM, 0 nM and the positive control for CFP. Error bars show the average deviation from the mean of the three measurements.

As a reason for the low fluorescence at high 3OC6-HSL concentrations you could also expect a possible toxicity for the cells, as low cell density would also mean low fluorescence. After looking at the OD₆₀₀ measurements (figure 5) this thought can be dismissed. The cells inoculated with 10 nM even grew a little better than the ones with 0.01 nM or 0 nM but still showed a less fluorescence.

Figure 5 Results of the OD₆₀₀ measurement of E.coli BL 21 J61002_BBa_J23100_BBa_ K1670005 pSB3C5_BBa_K1670001 induced with 3OC6-HSL concentrations of 0.01 nM, 10 nM, 0 nM and the positive control for CFP. Error bars show the average deviation from the mean of the three measurements.

References

1) Shong et al (2013) Directed Evolution of Quorum-Sensing Regulator EsaR for Increased Signal Sensitivity, ACS Chemical Biology 8 p.789-795
2) Shong et al (2013) Engineering the esaR Promoter for Tunable Quorum Sensing-Dependent Gene Expression, ACS Chemical Biology 2 p.568-575