Difference between revisions of "Part:BBa K2279001"
 
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[[file:T--TMMU-China--aimP.jpeg]]
 
[[file:T--TMMU-China--aimP.jpeg]]
  
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Then we inserted this gene to plasmid pSB1C3. We transformed this plasmid plays (one contains gene AimP) 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 AimP was success.  
+
Then we inserted this gene to plasmid pSB1C3. We transformed this recombinant plasmid (one contains gene AimP) into strain DH5α (<i>E. coli</i>). Then we picked some colonies for cultivation and extracted the recombinant plasmid, which was verified by PCR and sequencing. From the result of electrophoresis, we confirmed the transformation of AimP was success.  
  
 
[[file:T--TMMU-China--aimp4.jpeg]]
 
[[file:T--TMMU-China--aimp4.jpeg]]
  
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We then sequenced the positive bacteria and confirmed that the plasmid plays (one contains gene AimP) was indeed transferred to the bacteria. We expanded the bacteria and extracted plasmids from the bacteria. So far, we've successfully constructed AimP.
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===Biological Function===
 
===Biological Function===
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The B.subtilis bacteriophage phi3T employs the AimR-AimP QS system to make the lysis-lysogeny decision. AimP is the pre-pro-peptide. It is 43aa long. The mature signal peptide of AimP is SAIRGA. Binding of the mature signal peptide to 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.  
+
The <i>B.subtilis</i> bacteriophage phi3T employs the AimR-AimP QS system to make the lysis-lysogeny decision. AimP is the pre-pro-peptide. It is 43aa long. The mature signal peptide of AimP is SAIRGA. Binding of the mature signal peptide to 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.  
 
[[file:T--TMMU-China--aimfu.jpeg]]
 
[[file:T--TMMU-China--aimfu.jpeg]]
 
===Design===
 
===Design===
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By combining the expression of AimR and AimP components, we want to develop a synthetic QS system in B. subtilis for target gene autoinhibition.
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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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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.
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Figure 1. The design of autoinhibiton system
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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.
  
 
[[file:AimSR.jpeg]]
 
[[file:AimSR.jpeg]]
  
 +
Figure 2. The design of AimR-AimP based Sender and Receiver cells.
 
===Reference===
 
===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.
 
[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.
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 +
===Improvement by Evry_Paris-Saclay 2018===
 +
This part is not functional in ''Escherichia coli'' even after it has been codon-optimized for it ([[Part:BBa_K2675001|BBa_K2675001]]): when expressed in ''E. coli'' as the composite part [[Part:BBa_K2675041|BBa_K2675041]], it was not able to produce and release detectable levels the mature hexapeptide SAIRGA in the culturing media of ''E. coli'' cells.
 +
 +
This part has been improved to be functional in ''E. coli'': produce and secrete the mature arbitrium peptide of phage phi3T (SAIRGA). For this, the signal secretion of AimP was replaced by the OmpA secretion signal ([[Part:BBa_K2675002|BBa_K2675002]]). When expressed in ''E. coli'' as the composite part [[Part:BBa_K2675042|BBa_K2675042]], the mature hexapeptide SAIRGA was produced and released in the culturing media of ''E. coli'' cells.
  
  

Latest revision as of 09:23, 12 October 2018


AimP

AimP encodes a signal peptide.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 58
  • 1000
    COMPATIBLE WITH RFC[1000]

Plasmid construction

We used PCR to produce AimP gene fragment.

T--TMMU-China--aimP.jpeg

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

T--TMMU-China--aimp4.jpeg


Biological Function

The B.subtilis bacteriophage phi3T employs the AimR-AimP QS system to make the lysis-lysogeny decision. AimP is the pre-pro-peptide. It is 43aa long. The mature signal peptide of AimP is SAIRGA. Binding of the mature signal peptide to 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

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.

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.

Improvement by Evry_Paris-Saclay 2018

This part is not functional in Escherichia coli even after it has been codon-optimized for it (BBa_K2675001): when expressed in E. coli as the composite part BBa_K2675041, it was not able to produce and release detectable levels the mature hexapeptide SAIRGA in the culturing media of E. coli cells.

This part has been improved to be functional in E. coli: produce and secrete the mature arbitrium peptide of phage phi3T (SAIRGA). For this, the signal secretion of AimP was replaced by the OmpA secretion signal (BBa_K2675002). When expressed in E. coli as the composite part BBa_K2675042, the mature hexapeptide SAIRGA was produced and released in the culturing media of E. coli cells.