Difference between revisions of "Part:BBa K1025005"
 
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<partinfo>BBa_K1025005 short</partinfo>
 
<partinfo>BBa_K1025005 short</partinfo>
  
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iGEM trp sensor performance test part is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for performance test of our novel tryptophan sensor.  
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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===
 
===Usage and Biology===
  
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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.   
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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 ''lac''Z 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===
 
===Functional Parameters===
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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.
 
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.
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[[File:sensor test.jpg|300px|thumb|left]]
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[[File:sensor test.jpg|375px|thumb|center]]
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== Characterization by 2022 Canton_HS ==
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J23119 pro-CueR- rrnB T1 ter- T7Te ter- CopA pro-amilGFP- rrnB T1 ter- T7Te ter
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== Profile ==
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Name: CuR-CopA-amilGFP
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Base Pairs: 1703 bp
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Origin: E.coli, genome
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Properties: Monitor the Cu concentration
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== Usage and biology ==
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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.
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[[File:T--Canton HS--BBa K4291016-figure 11.jpg|500px|thumb|center|Figure 1. map of pUC19-CuR-CopA-amilGFP]]
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The components of this plasmid are described as follows:
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==BBa_K4291012 ==
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Name: CueR
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Base Pairs: 408 bp
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Origin: E.coli, genome
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Properties: regulate the cooper efflux
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== Usage and biology ==
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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.
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== BBa_K4291013 ==
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Name: CopA promoter
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Base Pairs: 287 bp
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Origin: E.coli, genome
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Properties: Cu+-transporting ATPase pump
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== Usage and biology ==
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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.
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Latest revision as of 10:07, 13 October 2022

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.

Sensor test.jpg

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.

Figure 1. map of pUC19-CuR-CopA-amilGFP

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3394
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3394
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3394
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3394
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3394
  • 1000
    INCOMPATIBLE 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).