Difference between revisions of "Part:BBa K5068012"
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    <h2>BBa_K5068012 (pET28a-RppA)</h2>
 
  
 
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Revision as of 08:04, 29 September 2024


RppA

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Construction Design

The pET28a RppA is composed of BBa_K5068011 (RppA) and BBa_K3521004 (pet28a backbone). We used NCBI to locate the gene sequence of RppA, connected RppA to the vector through homologous recombination (Fig. 1), and then transferred it into E. coli DH5α for copying.

Figure 1: The map of pET28a-RppA
Fig. 1. The map of pET28a-RppA

Engineering Principle

In order to visualize the more efficient expression of genes, we also selected a reporter gene 1,3,6,8-tetrahydroxynaphthalene synthase (RPPA). Reported studies have reutilized type III polyketide synthase (PKS) 1,3,6,8-tetrahydroxynaphthalene synthase (THNS); RppA (also known as RppA) developed a simple and powerful enzyme-coupled malonyl-CoA biosensor. RppA is responsible for the conversion of malonyl-CoA to 1,3,6,8-tetrahydroxynaphthalene (THN) and then to flavomycin (Fig 2). Because flavin is red, it can be used as a direct colorimetric indicator of intracellular malonyl-CoA levels. Therefore, we draw on the reporter gene of the sensor[1].

Figure 2: A type III PKS RppA can be repurposed as a malonyl-CoA biosensor.
Fig. 2. A type III PKS RppA can be repurposed as a malonyl-CoA biosensor.

Experimental Approach

The plasmid pET28a-RppA was constructed by homologous recombination method and transformed into E. coli DH5α strains (Fig 3.A). The agarose gel electrophoresis image from monoclonal PCR using single colonies from the transformation plates shows distinct bands corresponding to the fragments RppA (Fig 3.B). This indicates that the recombinant plasmid has been successfully constructed and transformed into competent cells. Additionally, sequencing alignment results confirm that the gene sequences in the constructed pET28a-RppA plasmid match the designed sequences perfectly, with no mutations detected (Fig 3.C).

Figure 3: Plasmid transformation and transformant validation results. The length of RppA is 1158bp.
Fig. 3. Plasmid transformation and transformant validation results. The length of RppA is 1158bp.

Characterization

We transformed the plasmid pET-RppA into E. coli BL21 (DE3) and validated it. We culture the bacterial solution at OD600 to 0.6 and induce with IPTG at 16 degrees. After centrifugation, the OD600 of the supernatant solution was measured, and the absorbance of OD600 can characterize the expression level of the protein. In Fig. 4A, the blue strain can be seen. In Fig. 4B, LB serves as the control group and the blue solution can be seen in the RppA. The Fig. 4C shows that the OD600 of the experimental group (RppA) is significantly higher than that of the CK group, indicating RppA protein expression.

Figure 4: The RppA protein expression and OD600
Fig. 4. The RppA protein expression and OD600

Reference

1. Dongsoo Y, Jun W K, Min S Y, et al. Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(40): 9835-9844.