Difference between revisions of "Part:BBa K2279003"
 
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[[file:T--TMMU-China--papR.jpeg]]
 
[[file:T--TMMU-China--papR.jpeg]]
  
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Then we inserted this gene to plasmid pSB1C3. We transformed this plasmid (one contains gene PapR) into strain DH5α (<i>E. coli</i>). Then we picked some colonies for cultivation and extracted the plasmid, which was verified by PCR. From the result of electrophoresis, we confirmed the transformation of PapR was success.  
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Then we inserted this gene to plasmid pSB1C3. We transformed this plasmid (one contains gene PapR) into strain DH5α (<i>E. coli</i>). Then we picked some colonies for cultivation and extracted the plasmid, which was verified by PCR and sequencing. From the result of electrophoresis, we confirmed the construction of pSB1C3-PapR was succeeded.  
  
 
[[file:T--TMMU-China--papr1.jpeg]]
 
[[file:T--TMMU-China--papr1.jpeg]]
  
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We then sequenced the positive bacteria and confirmed that the plasmid plays (one contains gene PapR) was indeed transferred to the bacteria. We expanded the bacteria and extracted plasmids from the bacteria. So far, we've successfully constructed PapR.
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===Biological function===
 
===Biological function===
 
<i>B. cereus</i>. 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. PapR is 48 amino acids long and contains an N-terminal signal peptide that targets it for secretion. Outside the cell, the PapR pro-AIP is processed by the secreted neutral protease B (NprB) to form the active AIP. The mature PapR oligopeptide sequence is ADVPFEL.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.
 
<i>B. cereus</i>. 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. PapR is 48 amino acids long and contains an N-terminal signal peptide that targets it for secretion. Outside the cell, the PapR pro-AIP is processed by the secreted neutral protease B (NprB) to form the active AIP. The mature PapR oligopeptide sequence is ADVPFEL.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.
  
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[[File:PlcR.jpeg]]
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[[File:PlcR.jpeg|700px]]
  
 
Figure 1. PlcR activation Model, cited from reference [1].
 
Figure 1. PlcR activation Model, cited from reference [1].
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Figure 2. The design of PlcR-PapR based autoinduction system
 
Figure 2. The design of PlcR-PapR based autoinduction system
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Furthermore, we will develop a synthetic communication pathway between <i>B. subtilis</i> strains by co-culturing PapR-producing “sender” cells with PlcR-sensing “receiver” cells to induce gene expression.
 
Furthermore, we will develop a synthetic communication pathway between <i>B. subtilis</i> strains by co-culturing PapR-producing “sender” cells with PlcR-sensing “receiver” cells to induce gene expression.
  

Latest revision as of 02:17, 2 November 2017


PapR

PapR encodes a signal peptide. The sequence of mature PapR signal peptide is ADVPFEL. PapR binds to the transcription factor PlcR. 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 PapR gene fragment.

T--TMMU-China--papR.jpeg

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

T--TMMU-China--papr1.jpeg


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. PapR is 48 amino acids long and contains an N-terminal signal peptide that targets it for secretion. Outside the cell, the PapR pro-AIP is processed by the secreted neutral protease B (NprB) to form the active AIP. The mature PapR oligopeptide sequence is ADVPFEL.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.

PlcR.jpeg

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.

PlcAuto.jpg

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.

PlcSR.jpeg

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.