Difference between revisions of "Part:BBa K3718005"

 
 
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<partinfo>BBa_K3718005 short</partinfo>
 
<partinfo>BBa_K3718005 short</partinfo>
  
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The protein Modular Receptor Platform (MRP) is designed according to the paper published in 2017 (Chang et al.). When the antigen/ligand binds to the nanobody, dimerization of the protein CadC domains occur. As a result, this allows the binding of the pCadBA operon region and transcription downstream is promoted at the same time. The VHH region has high flexibility as it can be customized for binding with different targets, so that any specific targets with single-domain antibodies available will be able to bind with the VHH region for detection. The VHH used this part is anti-caffeine.
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https://2021.igem.org/wiki/images/thumb/6/63/T--Hong_Kong_UCCKE--MRP.png/800px-T--Hong_Kong_UCCKE--MRP.png
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<p>
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We aim to test for the expression of the Modular Receptor Platform (MRP) under some control. By including the Lac operon promoter, it is simulated that downstream genes are expressed under the control of one operon-promoter. <i>mrfp</i> works as a reporter gene. It is expected that the MRP and mRFP will express in <i>E. coli</i> under IPTG induction.
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<h1>Characterization</h1>
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<h2>Transformation and expression in two strains of <i>E. coli</i></h2>
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<p>
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The recombinant plasmid containing K3718005 is synthesized by IDT.
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</p>
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<p>
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The plasmid is transformed into DP5a and BL21. DH5a is an <i>E. coli</i> strain, which maximizes transformation efficiency, while BL21 is an expression strain. Since we had encountered difficulties in verifying transformation via cPCR in BL21, team CityU_HK helped us with the following steps.
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<h3>Conformation of transformation via cPCR:</h3>
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https://2021.igem.org/wiki/images/thumb/d/d0/T--Hong_Kong_UCCKE--cPCR_MRP.png/800px-T--Hong_Kong_UCCKE--cPCR_MRP.png
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<p>
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The results show that the plasmid has been successfully transformed into BL21 and DH5a <i>E .coli</i> as the amplified region is 2.6kb. Both colonies’ band size is between 3000kb and 2000kb, showing that the plasmid is successfully transformed.
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<h2>Induced expression</h2>
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<p>
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To ensure the expression of MRP, the <i>mrfp</i> gene is added downstream of MRP. Expression of mRFP allows a visible signal to be observed when the genes downstream of the lac operon are expressed by the <i>E. coli</i> through the addition of IPTG.
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</p>
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https://2021.igem.org/wiki/images/thumb/e/eb/T--Hong_Kong_UCCKE--induction_mic.png/800px-T--Hong_Kong_UCCKE--induction_mic.png
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<p>
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The images from two fields clearly show that mRFP is expressed when IPTG is added, which convinces us that there is a large chance that MRP is also expressed when the operon is activated. However, SDS-PAGE and western blotting are required to confirm the expression
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<partinfo>BBa_K3718005 parameters</partinfo>
 
<partinfo>BBa_K3718005 parameters</partinfo>
 
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<h3>References</h3>
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H.-J. Chang, P. Mayonove, A. Zavala, A. De Visch, P. Minard, M. Cohen-Gonsaud, and J. Bonnet, “A modular receptor platform to expand the sensing repertoire of bacteria,” ACS Synthetic Biology, vol. 7, no. 1, pp. 166–175, 2017.
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Latest revision as of 06:46, 17 October 2021


Device for testing the expression of MRP

The protein Modular Receptor Platform (MRP) is designed according to the paper published in 2017 (Chang et al.). When the antigen/ligand binds to the nanobody, dimerization of the protein CadC domains occur. As a result, this allows the binding of the pCadBA operon region and transcription downstream is promoted at the same time. The VHH region has high flexibility as it can be customized for binding with different targets, so that any specific targets with single-domain antibodies available will be able to bind with the VHH region for detection. The VHH used this part is anti-caffeine.

800px-T--Hong_Kong_UCCKE--MRP.png

We aim to test for the expression of the Modular Receptor Platform (MRP) under some control. By including the Lac operon promoter, it is simulated that downstream genes are expressed under the control of one operon-promoter. mrfp works as a reporter gene. It is expected that the MRP and mRFP will express in E. coli under IPTG induction.


Characterization

Transformation and expression in two strains of E. coli

The recombinant plasmid containing K3718005 is synthesized by IDT.

The plasmid is transformed into DP5a and BL21. DH5a is an E. coli strain, which maximizes transformation efficiency, while BL21 is an expression strain. Since we had encountered difficulties in verifying transformation via cPCR in BL21, team CityU_HK helped us with the following steps.

Conformation of transformation via cPCR:

800px-T--Hong_Kong_UCCKE--cPCR_MRP.png

The results show that the plasmid has been successfully transformed into BL21 and DH5a E .coli as the amplified region is 2.6kb. Both colonies’ band size is between 3000kb and 2000kb, showing that the plasmid is successfully transformed.

Induced expression

To ensure the expression of MRP, the mrfp gene is added downstream of MRP. Expression of mRFP allows a visible signal to be observed when the genes downstream of the lac operon are expressed by the E. coli through the addition of IPTG.

800px-T--Hong_Kong_UCCKE--induction_mic.png

The images from two fields clearly show that mRFP is expressed when IPTG is added, which convinces us that there is a large chance that MRP is also expressed when the operon is activated. However, SDS-PAGE and western blotting are required to confirm the expression


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 282
    Illegal BglII site found at 1156
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 848
    Illegal AgeI site found at 1750
    Illegal AgeI site found at 1862
  • 1000
    COMPATIBLE WITH RFC[1000]







References

H.-J. Chang, P. Mayonove, A. Zavala, A. De Visch, P. Minard, M. Cohen-Gonsaud, and J. Bonnet, “A modular receptor platform to expand the sensing repertoire of bacteria,” ACS Synthetic Biology, vol. 7, no. 1, pp. 166–175, 2017.