Team:Tsinghua/Parts Characterization
Characterization
Overview
As our expectation, the engineered bacteria could sense the C4-HSL signal derived from P . aeruginosa. Thus, a C4-HSL report system should be considered and established. Here we characterized the composite part BBa_K1893003 as the report system. Since this part was not distributed, it was synthesized in BGI QINGLAN BIOTECH according to the sequence offered on the website and then integrated into plasmid pSB1C3. (The backbone of pSB1C3 is derived from distributed part BBa_R0071)
Note: the Rhl and pRhl are WT and not improved with relatively lower expression level, compared with the part improved by iGEM 2014 Tokyo_Tech.
Test of C4-HSL report system in BL-21
Here we tested the working condition of 3 plasmids in E . Coli strain BL-21(DE3). The tested plasmid, pSYS, shares the same sequence with BBa_K1893003. For the other plasmids, pPRhl is derived from part BBa_R0071 and plasmid pSYS(LVA) is modified from pSYS (in which the Rhl protein is labeled with LVA tag), which serve as the control groups.
Note: LVA tag is used as degradation signal for cellular protein.
After transformation, the single colony was picked and inoculated into 2mL chloramphenicol LB to culture overnight at 37℃. Then 60 times diluted of the culture solution was prepared. After 60-90min of 220 rpm shake at 37℃, the OD600 of the solution reached 0.3-0.5. Then solution was separated into 3 groups (500 μL) and meanwhile different inducers (0.5 μL) were added. After 4 hours incubation at 37℃, the cell culture was aliguoted into 96-well plate, 100 μL per well, 4 wells per group. VARIOSCAN FLASH(TM) microplate reader was used to measure the RFU(Ex 490 nm, Em 525 nm). The value of each column(Relative RFU/Turbidity) was calculated by(RFU_exp/RFU_LB/OD600).
As the data illustrated below, the C4-HSL report system pSYS shows about 9-fold signal enhancement with 100μM C4-HSL and about 6-fold enhancement with 10μM C4-HSL(shown as Figure A). Another experiment was used to quantify the concentration of C4-HSL and the RFU(shown as Figure B).
Figure A Test of C4-HSL report system in BL-21. Plasmid were transformed into BL-21 and the value of Relative RFU/Turibidity was measured. The C4-HSL report system pSYS shows about 9-fold signal enhancement with 100μM C4-HSL and about 6-fold enhancement with 10μM C4-HSL.
Figure B Relative RFU/turbidity in C4-HSL gradient tested in BL-21. It is expected that this report system is able to response well to more than the threshold of 1μM C4-HSL.
Test of C4-HSL report system in different strains
Here we also tested the plasmid pSYS in other E . Coli strains including DH5ɑ and TOP-10, (shown as Figure C) indicating that the C4-HSL report system may have the highest efficiency in TOP-10 strain.(about 19 fold enhancement with 100μM C4-HSL and 13 fold enhancement with 10μM C4-HSL) The working condition of the report system in DH5ɑ is similar to the case in BL-21.
Figure C Test of C4-HSL report system in different strains.Each strains were tested in 3 parallel group with different treatment. The C4-HSL report system seems to have highest efficiency in TOP-10 strain and relatively lower efficiency in BL-21 and DH5ɑ.
Please refer to:
http://parts.igem.org/Part:BBa_R0071
http://parts.igem.org/Part:BBa_C0071
http://parts.igem.org/Part:BBa_K1893003
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