Part:BBa_K1025005
Tryptophan-sensor Part
Tryptophan-sensor Part is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for performance test of our novel tryptophan sensor.
Usage and Biology
The mechanism of this biosensor has been described in detail by Gong F. andYanofsky C.[1]. It was derived from one regulation sequence upstream of tryptophanase(tnaA) operon in wild type E.coli. This sequence codes one 24-residue nascent peptide. Following this nascent peptide sequence stands one transcription termination factor (Rho) recognition sites. When certain amount of tryptophan exists, it is recognized by the nascent peptide. This leads to the hindering of TnaC-peptidyl-tRNAPro from being cleaved from ribosome. This peptide-mRNA-ribosome complex blocks Rho factor’s access to its binding site which is just adjacent to termination codon of nascent peptide so that initiate the transcription of downstream sequence. As far as we know, this novel mechanism has not been utilized before as tryptophan sensor. Hence, as a proof of principle, we cloned beta-lactamase gene lacZ downstream of wild type nascent peptide and Rho interaction sequence.The assembly was cloned between the NcoI and BamHI sites of pTrc99A vector.
Functional Parameters
The plasmid was transferred into E.coli JM109. By measuring the activity of beta-lactamase activity after induction and 21-hours culture, we obtained our expected tryptophan dependent beta-lactamase activity increase with dynamic range up to 3mM tryptophan. The main results were shown as below.
Characterization by 2022 Canton_HS
J23119 pro-CueR- rrnB T1 ter- T7Te ter- CopA pro-amilGFP- rrnB T1 ter- T7Te ter
Profile
Name: CuR-CopA-amilGFP
Base Pairs: 1703 bp
Origin: E.coli, genome
Properties: Monitor the Cu concentration
Usage and biology
The plasmid was designed to monitor the cooper concentration using the amilGFP intensity. The CueR which belongs to mercury resistance regulator (MerR) family response the mental ions including cooper and silver. Meanwhile, the CueR control the expression of CopA promoter. In the MerR family, there are other metal-responsive metal-regulators including ZntR, CadR, CoaR which response to the heavy metal zinc, cadmium and cobalt, respectively. All of the metal-regulators has the same property that these regulators could balance the heavy metal ions in bacteria via regulating the transcription of heavy-metal-resistance genes.
The components of this plasmid are described as follows:
BBa_K4291012
Name: CueR
Base Pairs: 408 bp
Origin: E.coli, genome
Properties: regulate the cooper efflux
Usage and biology
BBa_K4291012 is an encoding sequence of cooper efflux regulator (CuR) which is sensitive to metal ions such as Cu+, Ag+, Au+. In addition, CuR controls the transcription of copA in response the cooper salt.
BBa_K4291013
Name: CopA promoter
Base Pairs: 287 bp
Origin: E.coli, genome
Properties: Cu+-transporting ATPase pump
Usage and biology
BBa_K4291013 is an encoding sequence copA which is a cooper-transporter P-type ATPase pump. The expression of copA is improved when exposed to metal ions such as silver and cupric ions due to eliminating the CuR effect.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3394
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3394
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3394
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3394
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3394
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3623
Reference
[1] Gong, F. & Yanofsky, C. Instruction of translating ribosome by nascent peptide. Science297, 1864-1867, doi:10.1126/science.1073997 (2002).
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