Lead repressor under control of constitutive promoter and strong RBS
Lead promoter repressor under the control of konstitutive promoter (K608002)
Usage and Biology
For our biosensor we use parts of the chromosomal lead operon of Cupriavidus metallidurans. Naturally the operon for lead resistance contains pbrT, which encodes a Pb(II) uptake protein, pbrA, which encodes a P-type Pb(II) efflux ATPase, pbrB, which encodes a predicted integral membrane protein and pbrC, which encodes a predicted prolipoprotein signal peptidase. The expression of the operon is regulated by the repressor pbrR. As a MerR like regulator, its stimulates the transcription of the operon in the presence of Pb2+ (Borremans et al., 2001). Our sensor system combines pbrRunder the control of a constitutive promoter and the pbrA promoter for the lead depending expression of sfGFP.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 465
Illegal NheI site found at 488
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
in vivo characterization
In addition to the other heavy metal sensors, we constructed a sensor for lead detection. It consists of the repressor PbrR which binds at the operator box downstream of the pbrAP promoter. The binding of the repressor is reversible in the presence of Pb2+ Ions. Those ions can weakened the repressors binding and hence, all genes downstream of the pbrAP promoter can be expressed. Like the former sensors this one encloses a sfGFP for detection via fluorescence. So if no lead is present in the media, the repressor binds to the operator box and the pbrAP promoter is blocked meaning that the transcription of sfGFP is prevented. No fluorescence signal is the results. By supplementation of lead, the repressor is separated from the operator box and the genes downstream of the promoter can be expressed.
The pbrAP promoter, the operator box and the PbrR repressor are parts of the chromosomal lead operon of Cupriavidus metallidurans (figure 1). This was cloned and transformed into E.coli KRX. This operon includes now the promoter pbrAP ( BBa_K1758332 )), which is regulated by the repressor PbrR. The PbrR belongs to the MerR family, of metal-sensing regulatory proteins, and is Pb2+-inducible. Our sensor system comprises pbrR ( BBa_K1758330 ) BBa_K1758330 ), which is under the control of a constitutive Promoter and pbrAP and a 5’ untranslated region, which controls the transcription of a sfGFP and increases the fluorescence. Fluorescence implemented by sfGFP protein is the measured output signal (figure 2 and figure 3).
The kinetic of our sensors response to different lead concentrations is shown in figure 2. The first 40 hours show a strong increase in fluorescence. After that the increase in fluorescence reaches a plateau. For better visualization the kinetics of figure 2 are represented as bars in figure 3. A fluorescence level difference for 60 min, 150 min and 650 min is represented.
The results of the lead sensor show no significant differences between the different concentrations (figure 3). This might be due to the pbrAP’s weak promoter strength in E. coli. Further reasons are most likely in the weak repressor binding to its operator. So, we suggest for the usage of this sensor, it has to be optimized. Moreover we were lacking time for further in vivo characterizations and different experimental setups. Hence, we did not use this sensor in further experiments regarding Cell-free-Protein-synthesis (CFPS). In the future a characterization in the CFPS systems would be desirable.
Borremans B., Hobman J. L., Provoost A., Brown N. L., van der Lelie D. (2001), Cloning and Functional Analysis of the pbr Lead Resistance Determinant of Ralstoniametallidurans CH34, J. Bacteriol., 183, 5651 – 5658