Part:BBa_K5068009

pET-op-merR-RppA

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2622
  Illegal NgoMIV site found at 2782
  Illegal NgoMIV site found at 4370
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction Design

The pET-op-merR-RppA is composed of BBa_K5068003 (op-merR), BBa_K5068011 (RppA) and BBa_K3521004 (pet28a backbone). We connected RppA to the vector through homologous recombination (Fig. 1) and then transferred it into E. coli DH5α for copying.

Fig. 1. The map of pET-op-merR-RppA

Engineering Principle

The core of our biosensor lies in the integration of metal-responsive transcriptional regulators, MerR with a pigment biosynthesis gene cluster in E. coli. MerR is a well-characterized regulator that responds to mercury ions (Jung, 2019; Hui, 2018). By linking these regulators to the genes responsible for producing pigments such as carotenoids or other naturally occurring colorimetric compounds, we can ensure that the biosensor exhibits a clear visual response upon exposure to these heavy metals (Kumar, 2017). The pronounced red color change associated with RppA could improve the visibility and sensitivity of detecting mercury (II). Therefore, incorporating RppA as a reporter may enhance the overall performance of the biosensor in terms of colorimetric response to the presence of mercury ions. The plasmid pET-op-merR-RppA corresponds to transcriptional activation mediated by Hg (II), enabling the expression of RppA in the presence of Hg (II). Upon transforming this plasmid into competent cells, a biosensor for Hg (II) detection can be established (Fig. 2).

Fig. 2. The principle of mercury detection

Experimental Approach

The plasmid backbone pET28a was linearized using inverse PCR. Subsequently, the genes op-merR and RppA were amplified via PCR, yielding three fragments necessary for vector construction (Fig. 3A). Each fragment had homologous arms introduced at both ends through the primers, which can be seen in Fig. 3A. Optimized plasmid pET-op-merR-RppA was transformed into E. coli TOP10 and BL21 strains using conventional 42°C heat-shock transformation. Transformation plates were incubated at 37°C for 12 h to 16 h and monoclonal cells were used for transformation validation in Figure 3B. Figure 3C sequencing results showed that the base sequence was consistent, indicating that the plasmid was successfully constructed.

Fig. 3. Transformation process and transformant validation. Vector pET-A: 2755bp; op-merR-A: 589bp; RppA-A: 1201bp.

Characterization

We transformed the plasmid into E. coli BL21 and coated it onto Hg²⁺ plates of different concentrations, without Hg²⁺ as the control group. We can see red colonies on the plates with 10 μmol Hg²⁺ and 20 μmol Hg²⁺, but the plates without mercury ions did not turn red. The red colonies were inoculated into the culture medium of different concentrations of Hg²⁺, and the absorbance A₂₄₅ was measured after centrifugation. According to Fig. 4A, the color gradually became red with the increase of Hg²⁺ concentration, and Fig. 4B showed that the A₂₄₅ of 5, 10, 20 μmol Hg²⁺ was significantly higher than that of 0 μmol Hg²⁺, indicating that RppA was expressed and the mercury detection system was sensitive.

Fig. 4. The effectiveness of mercury detection at different concentrations of Hg²⁺

References

Jung J, Lee SJ (2019) Biochemical and biodiversity insights into heavy metal ion-responsive transcription regulators for synthetic biological heavy metal sensors. J Microbiol Biotechnol 29(10):1522–1542. https://doi.org/10.4014/jmb.1908.08002
Kumar S, Verma N, Singh A (2017) Development of cadmium specific recombinant biosensor and its application in milk samples. Sensors Actuators B Chem 240:248–254
Hui CY, Guo Y, Yang XQ, Zhang W, Huang XQ (2018) Surface display of metal binding domain derived from PbrR on E. coli specifically increases lead(II) adsorption. Biotechnol Lett 40(5):837–845. https://doi.org/10.1007/s10529-018-2533-4