Designed by: natalie kuldell   Group: BE.109 Instructors BE.109 2006 MIT 20.109 Spring07 MIT 20.109 Fall07   (2005-12-14)

OmpR controlled mRFP

Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of mRFP (I13507). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. An alternative clone is M30012. Cells bearing this construct are pale pink.

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].


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.


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