Difference between revisions of "Part:BBa K4385012"

Revision as of 14:53, 11 October 2022

Copper sensor

The copper ion sensor part is composed of PcusC and CusSR two-component membrane associated sensor kinase system. CusSR is composed of membrane-associated histidine kinase CusS and transcription activator CusR, responsible for sensing the presence of external Cu2+.

Usage and Biology

When external copper is sensed, the transmembrane structure of CusS protein undergoes a conformational change, and at the same time, autophosphorylation occurs to transmit the signal into the cell. The phosphoryl group is then transferred to the CusR protein, triggering a conformational change in CusR, and the phosphoryl group binds to a sequence in front of the PcusC promoter to turn on downstream gene expression.

Characterization

To verify the function of Cu2+ inducible PcusC, we introduced GFP downstream of the promoter and transformed plasmid PCusC-GFP into the engineered strain XL1-Blue. Plasmid PCusC-GFP was constructed to pSB1C3 (in Fig.XX).

To verify the construction of PCusC-GFP, the digestion and agarose gel electrophoresis of PCusC-GFP were performed by a standard protocol and two fragments of 1024 bp and 2029 bp were obtained by the digestion with EcoR I and Pst I (in Fig.XX).

To verify copper inducible promoter, different concentrations of Cu2+ were added to the reaction system, and the response of the promoter to the concentration gradient of Cu2+ were reflected by detecting the change of fluorescence intensity. As shown in Fig.XX, the enhancement of fluorescence intensity with the increase of copper concentration and duration was evident as compared to the control group.

Figure 1. PCusC-GFP.

[[Image: Cu_sensor2.png|center|frame|40px|] Figure 2.Cu2+ induction of PCusC promoter.(A) Digestion and gel electrophoresis of PCusC-GFP. (B) Fluorescence intensity of GFP under different concentration of copper ions. The design construct was transformed into E. coli XL1-Blue. The Cu2+ was added when the OD600 value was reached 0.5. The fluorescence intensity was detected at indicated time and normalized by the OD600 value. *, P < 0.05 from control using Student’s t test..]

Sequence and Features