Regulatory
OmpR

Part:BBa_R0082

Designed by: Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)   Group: Antiquity   (2004-01-27)


Promoter (OmpR, positive)

Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.


Usage and Biology

In nature, this promoter is upstream of the ompC porin gene. The regulation of ompC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high osmolarity, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC.

Contribution: Hamburg 2016

Group: Hamburg / Authors: Kai Pohl, Daniel Wedemeyer

Summary: We analysed the noise produced by BBa_R0082 by flow cytometry using a ΔEnvZ strain.

In two approaches, we ligated BBa_R0082 (OmpR, positive promoter) with BBa_E0240 (GFP) and BBa_E0430 (eYFP) into pSB1C3 transformed both ΔEnvZ (JW3367-3) and wt (DH5a) E. coli strains with them. Constructs were validated by sequencing. We submitted the constructs as: BBa_K1909013 (Omp/GFP) and BBa_K1909014 (Omp/eYFP) to the registry.

Experiments

To determine the noise of the OmpR promoter, we measured the expression of our Fluorescence reporters by flow cytometry, comparing the ΔEnvZ (JW3367-3) to wt (DH5a) E. coli strain. Furthermore, we used JW3367-3 and DH5a strains without our reporter system as negative controls. Cells were incubated in LB medium to OD600 = 0.2, washed using 100 mM MgCl2 and diluted 1:100 prior to measuring.

Results

Negative controls (JW3367-3 and DH5a without reporter systems) showed only few to no fluorescence (Fig.1 A, B). GFP expression (Fig. 1 A) proved to be significantly higher in DH5a (green) cells as compared to EnvZ deficient JW3367-3 cells (pink). Although not as pronounced, this difference was also observable for eYFP expression in the respective cell lines (fig. 1 B). Differences in the expression of fluorescence reporters were confirmed by KS tests (GFP: p <= 0.001, eYFP: p <= 0.001).

BBa R0082-FACS noise.png

Leaky Expression by the OmpR-Regulated Promoter on Different Vectors

(Characterized by SDU-Denmark)

Leaky expression by the OmpR-regulated promoter is reduced when cloned into a low copy vector compared to a high copy vector.
The expression properties of the OmpR-regulated promoter were investigated using a reporter system containing RFP under control of the OmpR-regulated promoter, BBa_M30011, was cloned into E. coli strain SØ928 ΔompR, lacking the OmpR transcription factor, on a high copy vector. By using a ΔompR strain, the background generated by stimulation of the intrinsic OmpR system is removed, and the strain functions as a negative control.
RFP expression was assessed by fluorescence microscopy using an Olympus IX83 with a photometrics prime camera, with exposure time for RFP at 200 ms. Assessing the RFP expression by fluorescence microscopy, it was discovered that the OmpR-regulated promoter mediated gene expression even in the absence of its transcription factor, see Figure 1. This observation was confirmed by going through the literature [1].

T--SDU-Denmark--de-lyser-rodt.png

Figure 1. Fluorescence microscopy of RFP controlled by the OmpR-regulated promoter on a high copy vector in E. coli strain SØ928 ΔompR.


On the basis of this finding, controlled gene expression by the OmpR-regulated promoter required a low copy plasmid or insertion into the chromosome. Protein expression of RFP in pSB1C3 with a copy number of 100-300 plasmids per cell, and pSB3K3 with a copy number of 10-12 plasmids per cell, was studied by flow cytometry. As for the determination of noise levels in the weak, BBa_J23114, and strong BBa_J23102constitutive promoters, the experiment was carried out in both LB medium and M9 minimal medium, the latter supplemented with 0.2% glycerol. In the LB medium, selection was carried out by the addition of 30 µg/mL chloramphenicol, 30 µg/mL kanamycin, or 50 µg/mL ampicillin, depending on the resistance, and for M9 minimal medium, the concentrations used were 60 µg/mL chloramphenicol, 60 µg/mL kanamycin, and 100 µg/mL ampicillin. Excitation of RFP was at 561 nm, and emission was measured around 580 nm. Expression levels in both E. coli MG1655 and E. coli MG1655 ΔompR were studied to determine the baseline of the leaky expression not influenced by intrinsic pathways including the OmpR transcription factor.


T--SDU-Denmark--Flow-celle1-reg.png

Figure 2. Flow cytometric fluorescence measurements in arbitrary units as a function of time. Left: Cultures were grown in LB medium. Right: Cultures were grown in M9 minimal medium supplemented with 0.2% glycerol. Fluorescence of RFP expressed by the the OmpR-regulated promoter on the high copy vector, pSB1C3, and the low copy vector, pSB3K3, in MG1655 WT and ΔompR MG1655 strain. All fluorescence levels were measured relative to the negative control WT E. coli MG1655, and the weak and strong constitutive promoters are included as references. Standard error of mean is shown, but are in several cases indistinguishable from the graph.

Fluorescence levels in the two different media display similar behavior, as seen in Figure 2. The main difference observed, was that the decrease in fluorescence over time was faster in LB medium than in M9 minimal medium, in concordance with the observations made in previous experiments. On a general level, the data revealed, that MG1655 cloned with the POmpR-RFP reporter system on the high copy vector exhibited a fluorescence level, equivalent to that mediated by the strong constitutive promoter. On the low copy vector, the POmpR-RFP reporter system yielded a fluorescence level comparable to the gene expression mediated by the weak constitutive promoter. On the other hand, expression levels in the MG1655 ΔompR strain were markedly reduced compared to MG1655, indicating that pathways including the transcription factor OmpR interfere with RFP expression under these conditions. Again, the fluorescence levels observed for the POmpR-RFP reporter system on the low copy vector were distinctly lower than for the high copy vector.
All things considered, the OmpR-regulated promoter was found to exhibit leaky expression comparable to the expression levels mediated by the constitutive promoters. When cloned into a low copy vector, the leaky expression was reduced prominently. Thus, to obtain proper regulation of gene expression by the OmpR-dependent promoter, a low copy vector is required.

[1] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005;438(7067):441-2


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/prokaryote/ecoli
//classic/regulatory/uncategorized
//direction/forward
//function/coliroid
//promoter
//regulation/positive
//rnap/prokaryote/ecoli/sigma70
Parameters
biology
directionForward
negative_regulators
positive_regulators1