Difference between revisions of "Part:BBa K346000:Experience"
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We constructed a two-plasmid system to characterize the T3 polymerase and T3 promoters, using IPTG, as the inducer of T3 polymerase expression and GFP as the reporter gene. | We constructed a two-plasmid system to characterize the T3 polymerase and T3 promoters, using IPTG, as the inducer of T3 polymerase expression and GFP as the reporter gene. | ||
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Fig 1. The two-plasmid system we constructed to characterize T3 RNAP/T3 promoter pairs. T3 promoter was prefixed BBa_E0840 by insertion of annealing primers which would form EcoRI and SpeI sticky end. | Fig 1. The two-plasmid system we constructed to characterize T3 RNAP/T3 promoter pairs. T3 promoter was prefixed BBa_E0840 by insertion of annealing primers which would form EcoRI and SpeI sticky end. | ||
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Characterization of T3 promoters
We constructed a two-plasmid system to characterize the T3 polymerase and T3 promoters, using IPTG, as the inducer of T3 polymerase expression and GFP as the reporter gene.
Fig 1. The two-plasmid system we constructed to characterize T3 RNAP/T3 promoter pairs. T3 promoter was prefixed BBa_E0840 by insertion of annealing primers which would form EcoRI and SpeI sticky end. Induction temperature was 28-30℃
Induction time was 6-8h
IPTG concentration was 10^-3M; culture with no IPTG supplement as the negative control
Spin down the cells under 5000rpm, and discard the supernatant. Resuspend the pellet with PBS. The the intensity GFP was recorded by the microplate reader. Parameters were selected as suggested in BBa_E0840.
Figure 4: The strength of different T3 promoters under the same expression level of T3 polymerase (10^-3M IPTG). BBa_E0840 was combined with 11 T3 promoters from the genome of T3 phage. The x axis denotes strains that only differ in the T3 promoter strength, in accordance with the order from strong to weak. The Y axis denotes the GFP intensity normalized by OD600. We can see that these T3 promoters can be divided into 3 subgroups: weak, medium and strong.
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