Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Jianyi Huang
Date created: 2016-10-11 11:00:00
Date modified: 2016-10-17 08:49:31
pCon+RBS+tetR+DT+pT7+pTet+RBS+tetX-GFP(fusion protein)+RBS+T7RNAP+DT
| Types | DnaRegion |
| Roles | engineered_region Reporter |
| Sequences | BBa_K2150027_sequence (Version 1) |
Description
This part is a complete regulatory device sensitive to tetracycline. A fusion protein(referred to as tetX-GFP) of tetX(a tetracycline-degrading enzyme) and GFP combined with a 3*GGGGS linker is used to report the expression level of tetX. The first promoter(BBa_J23119) is constitutively on and can express tetR, a protein that can repress the downstream promoter pTet in the absence of tetracycline(Tc). As a result, no tetX-GFP is produced and no fluorescence can be detected. In the presence of Tc, however, tetR can be repressed by Tc and repression over pTet can be removed, resulting in the expression of tetX-GFP and T7RNAP. Under such circumstances, activity of tetX can be reported by green fluorescence and this activity can be amplified by T7RNAP-pT7 system.Notes
Linker for fusion protein is carefully designed to avoid repeated sequence which may cause problems in assembly.PT7 is placed upstream of pTet, regarding that pT7 is a strong promoter while pTet is relatively weak. Linker for fusion protein is carefully selected to avoid repeated sequence that may cause problems in assembly.
Source
Fusion protein: tetX is the same as (xxx), the protein linker is provided by our advisor Li Hua, and the GFP is from BBa_E0040. We combine this three parts using overlap extension polymerase chain reaction (OE-PCR).pT7:PCR amplified from BBa_K525998
T7RNAP:PCR amplified from genome of BL21.