Difference between revisions of "Part:BBa K3425100"

 
 
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BioBrick created from the modular nanobody-based biosensor published by Chang et al (2018). It's composed (from N-ter to C-ter) of the E. coli CadC DNA binding domain, a juxtamembrane bridge region, an artificial 16L transmembrane region and the caffeine-binding camellid nanobody. The nanobody binds the target in a way that the protein CadC domains dimerize, which allows for the binding of the pCadBA operon region and promotes transcription downstream.  
 
BioBrick created from the modular nanobody-based biosensor published by Chang et al (2018). It's composed (from N-ter to C-ter) of the E. coli CadC DNA binding domain, a juxtamembrane bridge region, an artificial 16L transmembrane region and the caffeine-binding camellid nanobody. The nanobody binds the target in a way that the protein CadC domains dimerize, which allows for the binding of the pCadBA operon region and promotes transcription downstream.  
 
The nanobody domain can be exchanged in order to bind to other targets. This gives this protein flexibility to choose potentially any target that has single domain antibodies available. The native CadC protein of E. coli is unlikely to promote unspecific transcription of pCadBA, but avoid using growth media with acid pH or high concentrations of lysine.
 
The nanobody domain can be exchanged in order to bind to other targets. This gives this protein flexibility to choose potentially any target that has single domain antibodies available. The native CadC protein of E. coli is unlikely to promote unspecific transcription of pCadBA, but avoid using growth media with acid pH or high concentrations of lysine.
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<h2>Transformation and expression of MRP gene in two strains of E. coli (Team: Hong_Kong_UCCKE 2021)</h2>
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<p>The recombinant plasmid containing K3718005 is synthesized by IDT.</p>
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https://2021.igem.org/wiki/images/2/2a/T--Hong_Kong_UCCKE--MRP_Exp_part.png
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<p>The plasmid is transformed into DP5a and BL21. DH5a is an <i>E. coli</i> strain which maximises transformation efficiency, while BL21 is an expression strain.</p>
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<p>Since we had encountered difficulties in verifying transformation via cPCR in BL21, team CityU_HK helped us with the following steps.</p>
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<h4>Confirmation of transformation via cPCR:</h4>
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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>The results show that the plasmid has been successfully transformed into BL21 and DH5a as the amplified region is 2.6kb. Both colonies’ band size is between 3000kb and 2000kb, showing that the plasmid is successfully transformed as a result.</p>
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<h4>Expression of MRP:</h4>
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<p>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 <i>E. coli</i> through the addition of IPTG.</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>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. This shows the MRP can be probably be expressed under a inducible promoter, in future context of the CadBA promoter. However, SDS-PAGE and western blotting are required to confirm the expression.</p>
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Latest revision as of 13:24, 20 October 2021


DNA Binding Domain-caffeine nanobody fusion protein

BioBrick created from the modular nanobody-based biosensor published by Chang et al (2018). It's composed (from N-ter to C-ter) of the E. coli CadC DNA binding domain, a juxtamembrane bridge region, an artificial 16L transmembrane region and the caffeine-binding camellid nanobody. The nanobody binds the target in a way that the protein CadC domains dimerize, which allows for the binding of the pCadBA operon region and promotes transcription downstream. The nanobody domain can be exchanged in order to bind to other targets. This gives this protein flexibility to choose potentially any target that has single domain antibodies available. The native CadC protein of E. coli is unlikely to promote unspecific transcription of pCadBA, but avoid using growth media with acid pH or high concentrations of lysine.

Transformation and expression of MRP gene in two strains of E. coli (Team: Hong_Kong_UCCKE 2021)

The recombinant plasmid containing K3718005 is synthesized by IDT.

T--Hong_Kong_UCCKE--MRP_Exp_part.png

The plasmid is transformed into DP5a and BL21. DH5a is an E. coli strain which maximises 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.

Confirmation 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 as the amplified region is 2.6kb. Both colonies’ band size is between 3000kb and 2000kb, showing that the plasmid is successfully transformed as a result.

Expression of MRP:

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. This shows the MRP can be probably be expressed under a inducible promoter, in future context of the CadBA promoter. 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 56
    Illegal BglII site found at 930
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 622
  • 1000
    COMPATIBLE WITH RFC[1000]