Difference between revisions of "Part:BBa K2279000"

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[[file:T--TMMU-China--aimr1.jpeg]]
 
[[file:T--TMMU-China--aimr1.jpeg]]
  
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Then we inserted this gene to plasmid pSB1C3. We transformed this recombinant plasmid (one contains gene AimR) into strain DH5α (<i>E. coli</i>). Then we picked some colonies for cultivation and extracted the recombinant plasmid, which was verified by enzyme digestion using PstI and EcoRI enzymes. From the result of electrophoresis, we confirmed the construction of pSB1C3-AimR was succeeded.  
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Then we inserted this gene to plasmid pSB1C3. We transformed this recombinant plasmid (one contains gene AimR) into strain DH5α (<i>E. coli</i>). Then we picked some colonies for cultivation and extracted the recombinant plasmid, which was verified by enzyme digestion using PstI and EcoRI enzymes and sequencing. From the result of electrophoresis, we confirmed the construction of pSB1C3-AimR was succeeded.  
  
 
[[file:T--TMMU-China--aim2.jpeg]]
 
[[file:T--TMMU-China--aim2.jpeg]]
  
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We then sequenced the positive bacteria and confirmed that the plasmid plays (one contains gene AimR) was indeed transferred to the bacteria. We expanded the bacteria and extracted plasmids from the bacteria. So far, we've successfully constructed AimR.
 
  
 
===Biological function===
 
===Biological function===
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===Design===
 
===Design===
 
By combining the expression of AimR and AimP components, we want to develop a synthetic QS system in <i>B.subtilis</i> for target gene autoinhibition.
 
By combining the expression of AimR and AimP components, we want to develop a synthetic QS system in <i>B.subtilis</i> for target gene autoinhibition.
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[[file:AimAuto.jpeg]]
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[[file:AimAuto.jpeg|500px]]
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Figure 1. The design of  autoinhibiton system
  
 
A synthetic communication pathway between <i>B.subtilis</i> strains by co-culturing AimP-producing “sender” cells with AimR-sensing “receiver” cells to inhibit gene expression was also designed.
 
A synthetic communication pathway between <i>B.subtilis</i> strains by co-culturing AimP-producing “sender” cells with AimR-sensing “receiver” cells to inhibit gene expression was also designed.
  
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[[file:AimSR.jpeg]]
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[[file:AimSR.jpeg|700px]]
  
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Figure 2. The design of AimR-AimP based Sender and Receiver cells.
  
 
===Characterization===
 
===Characterization===
 
We constructed the pDG1730-pAim-AimR-AimP-pAimX-GFP (abbreviated as pDG1730-Aim-GFP), then we transformed this plasmid into <i>B.subtilis</i> 168 strain and knocked this composite part into the <i>amyE</i> locus. We also incorporated the pDG1730 vector into the B. subtilis 168 strain. Compared to this reference strain, we found that the pAim-AimR-AimP-pAimX-GFP composite part knocked-in strain can emit green fluorescence.
 
We constructed the pDG1730-pAim-AimR-AimP-pAimX-GFP (abbreviated as pDG1730-Aim-GFP), then we transformed this plasmid into <i>B.subtilis</i> 168 strain and knocked this composite part into the <i>amyE</i> locus. We also incorporated the pDG1730 vector into the B. subtilis 168 strain. Compared to this reference strain, we found that the pAim-AimR-AimP-pAimX-GFP composite part knocked-in strain can emit green fluorescence.
  
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[[file:Aim-fluorescence副本.jpg|500px|thumb|left]]
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[[file:Aim-fluorescence副本.jpg|700px]]
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Figure 3. Characterization of the pAim-AimR-AimP-pAimX-GFP composite part
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To explore the dynamics of the auotoinhibition of the synthetic AimR-AimP system, we track the green fluorescence intensity of the pAim-AimR-AimP-pAimX-GFP knock-in strain along time using the Microplate Reader. As shown in the Figure 9, we found that the expression of GFP increased steadily to a peak, and then the green fluorescence intensity decreased and finally reached a plateau. This result is consistent with the autoinhibition prediction of the pAim-AimR-AimP-pAimX-GFP composite part.
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[[File:Aimcurve.jpg|700px]]
  
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<p>  </p>
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Figure 4.Fluorescence curve of the pAim-AimR-AimP-pAimX-GFP composite part along time
  
 
===Reference===
 
===Reference===

Latest revision as of 02:18, 2 November 2017


AimR

AimR is a transcription factor.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 429
    Illegal SapI.rc site found at 169
    Illegal SapI.rc site found at 492

Plasmid construction

We used PCR to produce AimR gene fragment.

T--TMMU-China--aimr1.jpeg

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

T--TMMU-China--aim2.jpeg


Biological function

The B.subtilis bacteriophage phi3T encode the Aim system to make lysis-lysogeny decision. The AimR is a transcription factor, the AimP is the propeptide of the mature signal peptide. However, binding of AimP to the AimR will disrupt the dimer forms of AimR. After that, the AimR can no longer bind to the promoter of AimX, a potential non coding RNA involved in the process of lysis-lysogeny. T--TMMU-China--aimfu.jpeg

The AimR-AimP system and its role in the phage lysis-lysogeny decision, cited from reference [1].

Design

By combining the expression of AimR and AimP components, we want to develop a synthetic QS system in B.subtilis for target gene autoinhibition. AimAuto.jpeg

Figure 1. The design of autoinhibiton system

A synthetic communication pathway between B.subtilis strains by co-culturing AimP-producing “sender” cells with AimR-sensing “receiver” cells to inhibit gene expression was also designed.

AimSR.jpeg

Figure 2. The design of AimR-AimP based Sender and Receiver cells.

Characterization

We constructed the pDG1730-pAim-AimR-AimP-pAimX-GFP (abbreviated as pDG1730-Aim-GFP), then we transformed this plasmid into B.subtilis 168 strain and knocked this composite part into the amyE locus. We also incorporated the pDG1730 vector into the B. subtilis 168 strain. Compared to this reference strain, we found that the pAim-AimR-AimP-pAimX-GFP composite part knocked-in strain can emit green fluorescence.

Aim-fluorescence副本.jpg

Figure 3. Characterization of the pAim-AimR-AimP-pAimX-GFP composite part

To explore the dynamics of the auotoinhibition of the synthetic AimR-AimP system, we track the green fluorescence intensity of the pAim-AimR-AimP-pAimX-GFP knock-in strain along time using the Microplate Reader. As shown in the Figure 9, we found that the expression of GFP increased steadily to a peak, and then the green fluorescence intensity decreased and finally reached a plateau. This result is consistent with the autoinhibition prediction of the pAim-AimR-AimP-pAimX-GFP composite part. Aimcurve.jpg

Figure 4.Fluorescence curve of the pAim-AimR-AimP-pAimX-GFP composite part along time

Reference

[1] Erez, Z., Steinberger-Levy, I., Shamir, M., Doron, S., Stokar-Avihail, A., Peleg, Y., Melamed, S., Leavitt, A., Savidor, A., Albeck, S., et al. (2017). Communication between viruses guides lysis-lysogeny decisions. Nature 541, 488-493.