Team:BFSUICC-China/Design

BFSUICC-China

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Description

Desgin Experiments Protocol Interlab Model Results Demonstrate Improve Attributions

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Design

We aim to detect the concentration of copper ion in  the water. According to our goal, we design a circuit  
 which can improve previous copper detection  system to make it efficient and  intelligent.

 

Circuit

The design of BBa_K2555002

We design two basic parts to form a whole system, sensor part and regulator part:

 

 

 

Sensor Part

The design of BBa_K2555000 (optimum BBa_K1555000)

 

 

Previous part BBa_K1555000 in the registry

 

Escherichia coli has high tolerance for copper. CopA is the central component of copper homeostasis in E. coli, which removes Copper ions from the cytosol into the periplasm (Stoyanov et al .,2001). CopA is required for intrinsic copper resistance (Outten et al.,2000). The promoter of cop A is more sensitive to copper than the other two copper-responsive promoter. So, we use this promoter in our sensor part. Moreover, we add RiboJ between promoter and RBS of previous Part BBa_K1555000. RiboJ is an insulator commonly used in genetic circuits to prevent unexpected interactions between neighboring parts. Insulation with RiboJ may increase protein abundance. We replace GFP to sfGFP. The investigation has found that sfGFP increased resistance to denaturation, improved folding kinetics, increased resistance to aggregation during refolding(Andrews et al., 2007), and was very useful for improved protein detection(Cabantous et al., 2006).

 

Regulator Part

The design of BBa_K2555002

In the absence of metal ,the metalloregulator CueR bends the DNA so that the RNA polymerase cannot interact with the pCopA promoter properly, thereby repressing transcription (Outten et al., 1999). Upon metal coordination, CueR enables the DNA to assume a conformation that can activate the transcription process. CueR can behave as a net activator or a net repressor under different copper concentrations through interaction with RNA polymerase.( Danya J et al., 2015). We link L-arabinose inducing promoter-pBAD and CueR together. We can control the amount of Cue R expression though adding different concentration of arabinose.

 

 

The design of BBa_K2555003

BBa_K2555003 is used to indirectly reflect the expression of cueR of BBa_K2555002 under different concentration of L-arabinose by measuring green fluorescent intensity with plate reader.

 

 

References:

Andrews BT, Schoenfish AR, Roy M, Waldo G and Jennings PA. The rough energy landscape of superfolder GFP is linked to the chromophore. J Mol Biol 2007;373: 476-490.

Cabantous S and Waldo G. In vivo and in vitro protein solubility assays using split GFP. Nat Methods 2006; 3: 845-854.

Outten, C.E., Outten, F.W., and O’Halloran, T.V. (1999). DNA distortion mechanism for transcriptional activation by ZntR, a Zn(II)-responsive MerR homologue in Escherichia coli. J. Biol. Chem. 274, 37517–37524.

Outten, F.W., Outten, C.E., Hale, J.A., and O’Halloran, T.V. (2000). Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal Mer homologue, CueR. J. Biol. Chem. 275, 31024-31029.

Philips, S.J., Canalizo-Hernandez M., Yildirim I., Schatz G., Mondragón A., O’Halloran T., (2015) "Allosteric transcriptional regulation via changes in the overall topology of the core promoter." Science 349: 877-881

Stoyanov, J.V., Hobman, J.L., and Brown, N.L. (2001). CueR (YbbI) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA. Mol. Microbiol. 39, 502–512