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
Sweet Pressure Part （
RBS34）
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K1025008_sequence (Version 1) |
Description
iGEM sweet pressure part 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 maltose hydrolase expression which functioned in a maltose-sole carbon source culture condition. It is achieved by cloning E. Coli maltose hydrolase gene (malQ) downstream of our previously constructed tryptophan biosensor which is controlled by the strict araBAD promoter in one malQ single deletion strain E. Coli JW3379.First of all, we tested the growth rate of JW3379 in M9 minimal culture medium (about the component of M9 minimal medium, please refer to our note) with glucose and maltose as single carbon source, respectively. The results showed that with the same initial condition (the seed bacterium were from a LB culture and the initial OD600 was both set to be 0.16) after culturing in 5mL medium for about 12h in a 15mL centrifuge tube, glucose M9 medium gave a final biomass concentration of 0.58 (OD600) compared with 0.20 in maltose single carbon source M9 medium. Although this result showed that we have space to obtain tryptophan dependent growth rate by finely tuning the culture condition, due to the leakage effect of our novel tryptophan biosensor, the first rounds of trial failed. Thus, we constructed a random ribosome biding sequence (RBS) library upstream of malQ and utilized strictly controlled araBAD promoter upstream of malQ in pTRc99A vector. We applied this random library to select for the biggest growth difference between maltose and glucose M9 minimal culture condition. After the first round of selection, four strains were picked out. We further tested its performance by measuring the growth rate in different concentration of tryptophan addition. One of them was RBS_3-4.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].MalQ was sythesized by DNA 2.0 corporation with codon optimization according to GenBank DQ019991.[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) |
| BAD prompter RBS_BAD tnaC Rho binding site RBS_3-4 malQ rrnB_ternimator | 1,304 305,316 317,397 398,604 605,617 618,2702 2847,3004 | promoter feature/promoter feature/rbs ribosome_entry_site CDS feature/cds non_covalent_binding_site feature/binding feature/rbs ribosome_entry_site CDS feature/cds engineered_tag feature/barcode |