Difference between revisions of "Part:BBa K346002:Experience"
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===Applications of BBa_K346002=== | ===Applications of BBa_K346002=== | ||
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| + | == '''Promoter Characterization''' == | ||
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By annealing PmerT-forward (5’-3’) and PmerT-reverse (5’-3’) primers, PTPCAD carrying a sticky end of EcoRI and SpeI was cloned upstream of BBa_E0840, a GFP generator. With exogenous expression of MerR in bacteria, GFP’s expression could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and the BBa_J23103 (constitutive promoter)-merR into pSB1A3. | By annealing PmerT-forward (5’-3’) and PmerT-reverse (5’-3’) primers, PTPCAD carrying a sticky end of EcoRI and SpeI was cloned upstream of BBa_E0840, a GFP generator. With exogenous expression of MerR in bacteria, GFP’s expression could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and the BBa_J23103 (constitutive promoter)-merR into pSB1A3. | ||
Revision as of 00:47, 24 October 2010
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Applications of BBa_K346002
Promoter Characterization
By annealing PmerT-forward (5’-3’) and PmerT-reverse (5’-3’) primers, PTPCAD carrying a sticky end of EcoRI and SpeI was cloned upstream of BBa_E0840, a GFP generator. With exogenous expression of MerR in bacteria, GFP’s expression could be induced by Hg (II) in a dose response manner. The PTPCAD-E0840 was then cloned into pSB3K3 backbone and the BBa_J23103 (constitutive promoter)-merR into pSB1A3.
Protocols for promoter characterization
1.An overnight culture of bacteria carrying the two plasmids pSB3K3 and pSB1A3 were grown in LB broth with ampicillin and kanamycin at 37°C was reactivated by diluting the culture in a ratio of 1:100 with fresh LB.
2.When OD600 reached 0.4-0.6, the bacteria was disposed to several EP tubes, each owning 500uL, and different dose of Mercuric chloride solution was spplied with 3 duplicates and the final concentration varied from 0 to 1.0E-6 mol/L. 100uL of the 500uL was added to the black-96-well plates for GFP intensity’s measurement and another 100uL was added to the transparent-96-well plates for OD600 measurement.
3.In-plate culture fluorescence and OD600 was recorded at 20min intervals from 0 to 275min and the GFP intensity of 20min before inducement was also measured. Temperature was constant at 37°C.
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