Difference between revisions of "Part:BBa K2279002"

Revision as of 14:30, 31 October 2017

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

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]

Plasmid construction

We used PCR to produce PlcR gene fragment.

Then we inserted this gene to plasmid pSB1C3. We transformed this plasmid plays (one contains gene PlcR) into strain DH5α (E. coli). Then we picked some colonies for cultivation and confirmed the transformation result by PCR. From the result of electrophoresis, we confirmed the transformation of PlcR was success.

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.

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.

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.

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.

@@ Line 11: / Line 11: @@
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K2279002 SequenceAndFeatures</partinfo>
+===Plasmid construction===
+We used PCR to produce PlcR gene fragment.
+[[file:T--TMMU-China--plcr.jpeg]]
+Then we inserted this gene to plasmid pSB1C3. We transformed this plasmid plays (one contains gene PlcR) into strain DH5α (E. coli). Then we picked some colonies for cultivation and confirmed the transformation result by PCR. From the result of electrophoresis, we confirmed the transformation of PlcR was success.
+[[file:T--TMMU-China--plcr2.jpeg]]
+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===