Difference between revisions of "Part:BBa K1025005"

Revision as of 16:46, 25 September 2013

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.

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).

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1025005 short</partinfo>
-iGEM trp sensor performance test is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for performance test of our novel tryptophan sensor.
+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.
+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===