"
Team:HKUST/Project/Results and Discussion
From 2010.igem.org
Experimental Results
Construction of AIP Receptor for Localization Test
A.P32 Promoter (SacI)-[RBS]-agrC-mCherry-(XmaI/SmaI)
We successfully constructed agrC fused with mCherry using PCR. Agarose gel electrophoresis showed correct size.
B.P32 Promoter (SacI)-[RBS] -agrC-plnB-(XhoI)-mCherry-(XmaI)
We successfully built this construct by first ligating agrC-plnB into pBluescript SK (+) and then inserting mCherry after agrC-plnB into the same plasmid. Both steps were confirmed by enzyme digestion. The final construct was further confirmed by DNA sequencing. In addition, after we transformed this plasmid into E. coli, colonies on the plate gave red florescence under UV light. Since the red signal is produced by the florescence protein mCherry, it suggests that our designed part was successfully constructed and expressed in E.coli cells.
Construction of AIP Receptor for Functionality Test
A. P32 promoter (SacI)-[RBS]-agrC-(KpnI)
B. P32 promoter (SacI)-[RBS]-agrC-plnB-(KpnI)
1. The chimeric receptor agrC-plnB was successfully constructed through PCR.
2. The two different receptor genes agrC and agrC-plnB that we got through PCR were successfully cloned into plasmid pBluescript KS (+). The figure below shows the gel electrophoresis picture after enzyme digestion confirmation of the ligation product. It was further confirmed by DNA sequencing.
3.agrC and agrC-plnB were transferred from pBluescript KS (+) to our shuttle vector pMG36e by cutting the two genes out of pBluescript and then ligating them into pMG36e respectively. FIG 2 and FIG 3 show the picture of gel electrophoresis after enzyme digestion of the two constructs. And they were finally confirmed by DNA sequencing.
C. (EcoRI)(XbaI)-plnA promoter-[RBS]-(XmaI/SmaI)-gusA-(NotI)(EcoRI)- P32 promoter
1. The forward primer (79bp) and the reverse primer (73bp) of plnA promoter were annealed and extended at 72℃. Products were checked by polyacrylamide gel electrophoresis. Semi-log graph was plotted to calculate the size of plnA promoter which was 10bp longer than expected.
2. gusA was amplified by PCR with PBI121 as the template. But ligation of gusA into pBluescript KS (+) was failed despite several trials.
Construction of RIP Production Cassette
A. P32 promoter [RBS]-DD13-RIP
B. P32 promoter [RBS]-Signal peptide+DD13-RIP
We have successfully cloned these two constructs into pBluescript SK (+) and confirmed them by enzyme digestion and DNA sequencing.
Construction for Secretion Test of Hybrid Inhibiting Peptides
A.P32 promoter [RBS]-flag tag+DD13-RIP
1. We successfully cloned Flag-tag+DD13-RIP into pBluescript SK (+) and confirmed them by enzyme digestion and DNA sequencing.
2. We have inserted [RBS]Flag-tag into pMG36e and confirmed by enzyme digestion and DNA sequencing.
