Difference between revisions of "Part:BBa K2279002"

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PlcR

PlcR is a transcription factor. PlcR is the receptor of the mature signal peptide of PapR. This part is also an improvement of the part (BBa_K354001).

Sequence and Features

Plasmid construction

We used PCR to produce PlcR gene fragment.

Then we inserted this gene to plasmid pSB1C3. We transformed this recombinant plasmid (one contains gene PlcR) into strain DH5α (E. coli). Then we picked some colonies for cultivation and extracted the plasmid, which was verified by PCR. From the result of electrophoresis, we confirmed the construction of pSB1C3-PlcR was succeeded.

We then sequenced the positive bacteria and confirmed that the plasmid plays (one contains gene PlcR) was indeed transferred to the bacteria. We expanded the bacteria and extracted plasmids from the bacteria. So far, we've successfully constructed PlcR.

Biological function

B. cereus cause acute diarrheal disease by the production and secretion of a variety of hemolysins, phospholipases, and toxins. The production of virulence factors is controlled by the PlcR-PapR QS system. PlcR is the receptor for the mature PapR signal peptide. The PlcR protein has two domains. The helix-turn-helix domain is involved in the DNA binding activity. The TPR domain is involved in signal peptide binding. Inside the cell, PapR binds to the transcription factor PlcR, and this causes conformational changes in the DNA-binding domain of PlcR, facilitates PlcR oligomerization, DNA binding, and regulation of transcription.

Figure 1. PlcR activation Model, cited from reference [1].

Design

To develop a synthetic QS system in B. subtilis for target gene autoinduction, we are going to combine the expression of PlcR and PapR components.

Figure 2. The design of PlcR-PapR based autoinduction system

Furthermore, we will develop a synthetic communication pathway between B. subtilis strains by co-culturing PapR-producing “sender” cells with PlcR-sensing “receiver” cells to induce gene expression.

Figure 3. The design of PlcR-PapR based Sender and Receiver Cells.

Reference

[1] Grenha, R., Slamti, L., Nicaise, M., Refes, Y., Lereclus, D., and Nessler, S. (2013). Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR. Proc Natl Acad Sci U S A 110, 1047-1052.