Part:BBa_K3718005
Device for testing the expression of MRP
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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 282
Illegal BglII site found at 1156 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 848
Illegal AgeI site found at 1750
Illegal AgeI site found at 1862 - 1000COMPATIBLE WITH RFC[1000]
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.
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.
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:
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.
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
Reference
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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