Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Weifan Liang
Date created: 2013-09-13 11:00:00
Date modified: 2015-05-08 01:08:45
Tryptophan-sensor Part
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K1025005_sequence (Version 1) |
Description
iGEM trp sensor performance part is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for performance test of our novel tryptophan sensor. 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-tRNA^Pro 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 with IPTG induction.By measuring the activity of beta-lactamase activity (the protocol has been described in detail in our note, please refer to it) after induction and 21-hours culture, we obtained our expected tryptophan dependent beta-lactamase activity increase with dynamic range up to 3mM tryptophan.Notes
The genes were cloned easily by PCR and connected to plasmid backbone by in-fusion cloning.Source
TanC and Rho were synthesized by DNA 2.0 corporation according to reference[1]. LacZ was cloned from E.coli genome.[1]Gong, F. & Yanofsky, C. Instruction of translating ribosome by nascent peptide. Science297, 1864-1867, doi:10.1126/science.1073997 (2002).
| Sequence Annotation | Location | Component / Role(s) |
| tac promoter RBS_tac tanC Rho binding site RBS_tnaA lacZ rrnB_ternimator | 1,62 62,72 81,155 156,362 363,375 376,3450 3539,3696 | promoter feature/promoter ribosome_entry_site feature/rbs CDS feature/cds feature/binding non_covalent_binding_site ribosome_entry_site feature/rbs CDS feature/cds feature/barcode engineered_tag |