Part:BBa_K1349002
CusR box
The CusR box allow the binding of the CusR regulator. In E. coli, the CusR/CusS two-component system induces expression of genes involved in metal efflux under condition of elevated Cu concentration. Phosphorylated CusR specifically bind to CusR-dependent promoters.
Validation of the CusR box
In E. coli, the CusR/CusS two-component system induces expression of genes involved in metal efflux under condition of elevated Cu or Ag concentrations. Phosphorylated CusR specifically bind to CusR-dependent promoters. The CusR box is the recognition sequence for the protein CusR.
We designed a Part corresponding to the CusR box to be inserted downstream of a promoter to allow transcriptional regulation of the downstream cloned gene. In common with many response regulators we expect over-expression of CusR to result in binding and so regulation of an upstream promoter. These effects are expected through bound CusR physically interacting with RNA polymerase.
How we tested the BBa_K1349002 part:
Insertion of the BBa_K1349002 part downstream of a promoter is expected to allow the binding of the CusR regulator in response to metal (Cu or Ag) availability in the medium. While different in their biological role and cellular targets, these metals share very similar chemical and ligand- binding properties. Consequently, the presence of multiple CusR-boxes would be able to bind multiple CurR regulators as so to titrate the intracellular pool of CusR.
To test so, we inserted the CusR box (BBa_K1349002) in a pLac vector (AmpR) downstream of the Lac dependent promoter, followed by the GFP coding sequence. The Lac promoter is leaky enough to allow GFP synthesis without the addition of inducer to the culture medium, attesting that the construction allow gene expression. As our final objective a fine-tuning of our modified CurR regulatory cascade, we also tested the effect of multiple CusR-boxes insertion in our assay (1 box, 2 boxes and 3 boxes). As a control, the Plac promoter was fused to the GFP coding sequence without any CusR box. Strains were cultivated at 37°C in M9 medium until they reached an OD600nm=0,4 to 0,6. Then, an aliquot of each culture was treated with a sub-lethal concentration of AgNO3 (5 μM, see reference Gudipaty SA et al., 2012) while a second untreated aliquot was used as a control. The two series of experiments presented here were performed twice and showed similar results.
- Growth was determined overtime by following the culture OD at 600 nm on a TECAN instrument (Figure A).
- GFP synthesis was quantified by measuring the OD at 530 nm on a TECAN instrument. Then, fluorescence was normalized by dividing the results with the OD at 600 nm (Figure B).
Figure A: The presence of cusR boxes on a pLac vector affects cell growth in the presence of a sub-lethal dose of AgNO3.
As seen on figure A, the introduction of a unique CusR box in our test vector (P-cusB1-GFP) has no effect on growth compared to the control vector (P-GFP), with or without Ag supplementation to the medium. At the opposite the presence of 2 CusR boxes (P-cusBx2-GFP) or 3 CusR boxes (P-cusBx3-GFP) in our test vector has a very strong effect on growth when AgNO3 is added to the medium.
Conclusion on figure A:
As seen on figure A, the CusR-box part that we designed (BBa_K1349002) works as expected. The insertion of multiple CusR-boxes on the pUC18 vector (2 or 3 boxes) has almost no effect on untreated cells growth, but it leads to a culture growth defect in the presence of Ag. This suggest that the BBa_K1349002 part is able to titrate the endogenous CusR regulator in the cells, preventing a correct stress response when E. coli is exposed to sub-lethal concentrations of Ag.
Figure B: The insertion of 2 or 3 CusR boxes downstream of the Plac promoter allows regulation of gene expression.
- As seen on figure B for the untreated samples (“Untreated”), the expression of GFP increases when CusR-boxes (BBa_K1349002) are inserted into the pUC18 expression plasmid. The effect is particularly strong with the presence of 2 boxes and 3 boxes.
- Addition of Ag to the medium (“+AgNO3”) seems to have a negative effect on gene expression compared to the untreated samples. Because in this experiment, treated and untreated samples are not in the same growth phase, we are planning to reproduce this experiment with a lower dose of Ag to avoid Ag toxicity.
Conclusion on figure B:
Our results show that the CusR-box part that we designed (BBa_K1349002) is functional, as it allows the transcriptional control of the downstream gene. Interestingly, our data suggest that the insertion of 2 or 3 CusR-boxes is optimal to allow the binding of the CusR regulator.
Reference
Gudipaty SA, Larsen AS, Rensing C, McEvoy MM. Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS. FEMS Microbiol Lett. 2012 May;330(1):30-7.
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 |