Part:BBa_K4061000
OmpF promoter, controlled by OmpR regulatory protein as part of the Two Component System in E. coli
OmpF promoter is a OmpR regulated promoter that is activated under low medium osmolarity in E. coli especially. This promoter is reciprocally regulated with OmpC promoter (Bba_R0082) depending on the medium osmolarity. OmpF promoter is preferentially expressed in low medium osmolarity. In low osmolarity, the kinase activity of EnvZ (a histidine kinase) is repressed, leading to low concentration of phosphorylated OmpR (OmpR~P). Lower concentration of OmpR~P positively regulates the promoter by binding to high affinity sites (F1, F2, F3). At increased concentration of OmpR~P which occurs at a higher osmolarity, the OmpR~P binds the low affinity F4 site on the promoter which acts a negative regulator and represses the expression of OmpF promoter.
Usage and Biology
This promoter natively regulates the OmpF porin protein which has a relatively large diameter of pore. This is preferentially expressed on E. coli membrane in times when the environment osmolarity is extremely low and the nutrients are scarce. This way, the organism ensures appropriate exchange of ions and nutrients in order to maintain a good osmotic balance when internal osmolarity also starts to drop.
Contribution: HKUST 2021
Summary
We characterised the OmpF promoter with (BBa_K4061000) and without its negative regulator (BBa_R0084) region. This part contains the negative regulator domain F4 and we compare its activity to BBa_R0084 which does not contain this domain, only the 3 positive regulator regions. We observe that the presence of the negative regulator allows a better control of the promoter and allows a "switch-like" response for an increasing osmolarity.
Experiments
We got the complete sequence of OmpF promoter (with negative regulator) from E. coli K12 strain genome. We ordered the whole sequence in tandem with the mRFP1 reporter via IDT. This gave us the part BBa_K4061000. In order to analyse the action of negative regulator we PCRed out the fragment right before the positive regulators in order to eliminate the negative regulator. The resulting sequence is BBa_R0084 OmpF promoter with mRFP1 reporter. Cells transformed with these two constructs were cultured in LB media with varying NaCl concentrations and the fluorescence intensities were compared at different media concentrations - final Na+ concentrations of 0.033M, 0.048M, 0.078M, 0.131M. 0.171M, 0.214M, 0.256M and finally 0.299M. Concentrations below 0.171M are considered as "low osmolarity" from the ideal LB medium recipe.
Results and Discussion
We observe a steeper decline close to wild-type level for mRFP1 regulated under OmpF promoter with the negative regulator. The OmpF promoter without the negative regulator reduces expression of reporter only 1.6 times at a high osmolarity while the promoter with negative regulator reduced expression to 2 times. BBa_R0084 (without F4) regulated mRFP1, however, has a much higher fluorescence intensity as compared to the reporter regulated under BBa_K4061000 (with F4).
Note: Data from the two constructs, despite being taken concurrently, is not plotted on the same graph due to the difference in the intensities. Separating the graphs allow better visualisation of the trend in the promoter activity.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |