Team:SIAT-SCIE/Composite Parts
BBa_K2472003
BBa_K2472004
BBa_K2472005
Regulation System: Tet-On system
Inducer: aTC
The system was designed to express the CAHSs in E.coli, and then express in metazoan cells.
BBa_K2472007
pTac-Dsup
This is the system with a Tac promoter, which can work with the lac operators. It can be induced by simply lactose in culture medium. We use this device to find out the relationship between Dsup expression and survival rate, more details are presented in modeling
Dsup Design:
Tardigrade-unique DNA-associated protein with RBS
Design Notes
Length: 1338bp
Primers for isolation the sequence from backbone pMV, with RBS in the fwd primer.
Source
The gene sequence was isolated from R. varieornatus, a species of tardigrade.
References
Hashimoto, Takuma, et al. "Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein." Nature Communications 2015.9(2015) 7:12808.
Dsup Experience
We used pMD19-T0-Amp-T1-Ptet-tetR as our expression plasmid. E. coli MG1655 bacteria transformed with the plasmids were grown in 2ml LB at 37℃ overnight. To express the protein, the bacteria was diluted until the cell had reached exponential phase. After addition of aTC to make a final concentration of 50ng/μl, the cells were grown for 12 hours. The cells are collected by centrifuging at 10000rpm for 5min. The cell pellet collected from 10ml of bacterial culture was resuspended in 2ml of lysis buffer (ingredients required) and sonified for 8min on ice, following a centrifuge at 10000rpm for 15min to remove insoluble debris. The results were shown on a 10% SDS-PAGE.
The cells after induction were then disposed with a dose gradient of UV radiation. A survival rate assay was carried out.
Ptet-tetR-RBS-Dsup Design
Regulatory system for Dsup (or other tardigrade-unique proteins) with RBS
Design Notes
Length: 728bp+1338bp+RBS
Constructed by ligation of Dsup sequence with RBS and double digested plasmid pMD19-T0-Amp-T1-Ptet-tetR. Primers for isolation the sequence from backbone pMD19.
Source
The operon sequence was isolated from E. coli strain DH1.
References
Matsumoto, Yuki, et al. "Bacterial Cells Carrying Synthetic Dual-Function Operon Survived Starvation." Journal of Biomedicine & Biotechnology 2011.12(2011):489265.
Ptet-tetR-RBS-Dsup Experience
We used pMD19-T0-Amp-T1-Ptet-tetR as our expression plasmid. E. coli MG1655 bacteria transformed with the plasmids were grown in 2ml LB at 37℃ overnight. To express the protein, the bacteria was diluted until the cell had reached exponential phase. After addition of aTC to make a final concentration of 50ng/μl, the cells were grown for 12 hours. The cells are collected by centrifuging at 10000rpm for 5min. The cell pellet collected from 10ml of bacterial culture was resuspended in 2ml of lysis buffer (ingredients required) and sonified for 8min on ice, following a centrifuge at 10000rpm for 15min to remove insoluble debris. The results were shown on a 10% SDS-PAGE.
The part was characterized by concentration gradient of aTC and time gradient of induction. The procedures above were repeated with 0, 12.5, 25ng/μl of aTC and 1.5, 3, 6 hours of induction respectively.
