Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Weifan Liang
Date created: 2013-09-14 11:00:00
Date modified: 2015-05-08 01:08:45
Bitter Defender Part（
RBS_B0032）
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K1025007_sequence (Version 1) |
Description
iGEM bitter pressure part(B0032) is a plasmid constructed by 2013 iGEM Tsinghua-E team which is used for providing of selection pressure for the evolution and enrichment of tryptophan overproduction microorganism phenotype. This selection pressure was based on the tryptophan dependent tetracycline antiporter expression which functioned in tetracycline culture condition. It is achieved by cloning E. Coli tetracycline antiporter gene (tetA) downstream of our previously constructed tryptophan biosensor which is controlled by tac promoter between NcoI and BamHI restriction sites in pTrc99A vector. We constructed three bitter pressure part by utilizing three different RBS upstream of tetA gene. We transformed these vectors into three previously engineered E. coli with different tryptophan productivity (unpublished data). The tryptophan productivity of these nine strains (three RBS and three previous productivities) was further confirmed by HPLC after culture in M9YE medium for nearly 25h after 0.1mM IPTG induction. Strains carrying our bitter pressure part with RBS B0032 showed good tryptophan dependent growth property within the first 15h after culture. Further, as the increase of tetracycline, the selection pressure increased and the growth rate of strains decreased. However, with increased selection pressure,it could be observed that with time window of about 15h, strains with higher tryptophan overproduction showed much higher growth rate with the growth of tryptophan non producer trp000 nearly inhibited by high concentration of tetracycline.Notes
The genes were cloned easily by PCR and connected to plasmid backbone by in-fusion cloning.Source
TnaC and Rho were sythesized by DNA 2.0 corporation according to reference[1]. RBS_B0032 was from the biobrick B0032. TetA was cloned from plasmid pCM110.[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 tnaC Rho binding site RBS_B0032 tetA rrnB_ternimator | 1,62 63,72 81,155 156,362 363,375 376,1575 1664,1821 | promoter feature/promoter ribosome_entry_site feature/rbs CDS feature/cds non_covalent_binding_site feature/binding feature/rbs ribosome_entry_site feature/cds CDS feature/barcode engineered_tag |