Part:BBa_K5068007

p15A-op-merR-bspA-pcpS

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 5125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1337
  Illegal BamHI site found at 3589
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3615
  Illegal NgoMIV site found at 11153
  Illegal NgoMIV site found at 11291
  Illegal AgeI site found at 1172
  Illegal AgeI site found at 2829
  Illegal AgeI site found at 3938
  Illegal AgeI site found at 5211
  Illegal AgeI site found at 5535
  
• 1000
  COMPATIBLE WITH RFC[1000]

New Improved Part: BBa_K5068007 (p15A-op-merR-bspA-pcpS)

Summary

The bapA and pcpS genes play important roles in the production and transport of pigments. The bapA gene encodes a protein involved in the synthesis of the blue pigment indigo (McNerney, 2017), while the pcpS gene encodes a PPTase that activates acyl carrier proteins, facilitating the effective transport of the synthesized pigment within the cell. These two genes typically work in synergy, with bapA synthesizing indigo and pcpS enhancing the transport and stability of the pigment, thereby increasing the bacteria's survival in specific environments (Xie, 2019). In a heterologous expression system, the combination of bspA (a single-module non-ribosomal peptide synthetase) and pcpS enables the successful biosynthesis of indigo in Escherichia coli, demonstrating their collaborative role in the pigment biosynthetic pathway. Based on BBa_K1420004 (merR), we constructed a new combination plasmid BBa_K5068007 (p15A-op-merR-bspA-pcpS). This plasmid can be used to detect Hg. In addition, we also performed sequence optimization to improve the sensitivity of mercury detection.

Construction Design

The p15A-op-merR-bspA-pcpS is composed of BBa_K5068003 (op-merR), BBa_M36245 (Pcad), BBa_K4605002 (bspA), BBa_K5068002 (pcpS), and BBa_K5067004 (p15A). We connected the gene to the vector through homologous recombination (Fig. 1), and then transferred it into E. coli DH5α for copying.

Fig. 1. The map of p15A-op-merR-bspA-pcpS

Engineering Principle

The plasmid p15A-op-merR-bspA-pcpS corresponds to transcriptional activation mediated by Hg (II), enabling the expression of indigo pigment in the presence of Hg (II). Upon transforming this plasmid into competent cells, a biosensor for Hg (II) detection can be established (Fig. 2). This biosensor utilizes the production of indigo pigment as a visual indicator, allowing for the straightforward identification of mercury contamination in various samples.

Fig. 2. The principle of mercury detection

Experimental Approach

The plasmid backbone p15A (BBa_K5068009) was linearized using inverse PCR. Subsequently, the genes op-merR-merTPCAD and bspA-pcpS were amplified via PCR, yielding three fragments necessary for vector construction (Fig. 3). Each fragment had homologous arms introduced at both ends through the primers, which can be seen in Fig. 3.

Fig. 3. Electropherogram of fragments required for vector construction. The length of p15A is 5061bp, the length of merR is 568bp, and the lengths of bspA and pcpS are 4643bp.

These three fragments were then joined using homologous recombination. Because each pair of adjacent fragments contained homologous sequences, a circular structure was ultimately formed, resulting in the recombinant plasmid p15A-op-merR-bspA-pcpS (Fig. 4A). The constructed recombinant plasmid was subsequently transformed into competent E. coli strains TOP10 and BL21 (Fig. 4B), and the transformants were verified. Results from monoclonal PCR validation (Fig. 4C) and sequencing alignment (Fig. 4D) demonstrated that the sequences were highly consistent with the target sequence, with no significant mutations or insertions/deletions observed, confirming the successful transformation of the plasmid into E. coli. Ultimately, the recombinant plasmid was obtained, along with positive clones containing the recombinant plasmid in both the TOP10 and BL21 strains.

Fig. 4. Transformation process and transformant validation

Characterization

We transformed the plasmid into E. coli BL21 and coated it onto Hg²⁺ plates of different concentrations, without Hg²⁺ as the control group. In Figure 5, we can see blue colonies on the plates with 10 μmol Hg²⁺ and 20 μmol Hg²⁺, but the plates without mercury ions did not turn blue. This indicates that our mercury detection sensor is working, while also detecting mercury ions.

Fig. 5. The effectiveness of mercury detection at different concentrations of Hg²⁺

Then the blue colonies were inoculated into the culture medium of different concentrations of Hg²⁺, and the absorbance A₆₀₀ was measured after centrifugation. According to Fig. 6A, the color gradually became blue with the increase of Hg²⁺ concentration, and Fig. 6B showed that the A₆₀₀ of 5, 10, 20 μmol Hg²⁺ was significantly higher than that of 0 μmol Hg²⁺, indicating that the indigo gene bspA-pcpS was expressed and the mercury detection system was sensitive.

Fig. 6. The effectiveness of mercury detection at different concentrations of Hg²⁺

References

1. McNerney MP, Michel CL, Kishore K, Standeven J, Styczynski MP (2019) Dynamic and tunable metabolite control for robust minimal-equipment assessment of serum zinc. Nat Commun 10(1):5514. https://doi.org/10.1038/s41467-019-13454-1
2. Xie Z, Zhang Z, Cao Z, Chen M, Li P, Liu W, Qin H, Zhao X, Tao Y, Chen Y (2017) An external substrate-free blue/white screening system in Escherichia coli. Appl Microbiol Biotechnol 101(9):3811–3820.