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Team:Tokyo Tech/Protocol
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== <font size=5>'''Protocol''' </font>== | == <font size=5>'''Protocol''' </font>== | ||
=== Reporter Assay === | === Reporter Assay === | ||
| + | [[Image:Wash.jpg|thumb|450px|right|figure Wash]] | ||
| + | [[Image:Transfer_Function.png|thumb|450px|right|figure Hill function]] | ||
To quantitatively determine the performances of operator parts in the reporter plasmid, the change of fluorescence intensities of the reporter strains in the various concentrations of IPTG were measured. Overnight cultures of reporter strains grown at 37℃ in LB medium containing appropriate antibiotics were diluted 1:100 in LB medium and were incubated at 37℃ as fresh cultures. After their OD600 reached 0.6, the fresh cultures were diluted 1:1 in various concentrations of IPTG [0μM, 1 μM, 10 μM, 20 μM, 50μM, 100 μM and 1000 μM]. The fresh cultures were incubated at 37℃ for 12 hours and every 2 hours, 200 μl of each culture was taken to 1.5 ml tube and washed. Cultures were centrifuged for 2 min at 9000g, and the supernatant was discarded with a pipette. The pellet at the bottom of the tube was dissolved in 200 μl of PBS and was centrifuged for 2 min at 9000g, and the supernatant was discarded with a pipette. The pellet at the bottom of the tube was dissolved in 200 μl of PBS. 150 μl of the culture was taken to 96-well plate and its fluorescence intensity was measured by fluorimeter (FLA). The measured fluorescence intensity was corrected by subtracting the background fluorescence, measured in control wells containing 150 μl of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600). A strain, transformed with PtetR-GFP, which expresses GFP constitutively, was used as a positive control. A strain, transformed with Promoter less-GFP which does not express GFP was used as a negative control. | To quantitatively determine the performances of operator parts in the reporter plasmid, the change of fluorescence intensities of the reporter strains in the various concentrations of IPTG were measured. Overnight cultures of reporter strains grown at 37℃ in LB medium containing appropriate antibiotics were diluted 1:100 in LB medium and were incubated at 37℃ as fresh cultures. After their OD600 reached 0.6, the fresh cultures were diluted 1:1 in various concentrations of IPTG [0μM, 1 μM, 10 μM, 20 μM, 50μM, 100 μM and 1000 μM]. The fresh cultures were incubated at 37℃ for 12 hours and every 2 hours, 200 μl of each culture was taken to 1.5 ml tube and washed. Cultures were centrifuged for 2 min at 9000g, and the supernatant was discarded with a pipette. The pellet at the bottom of the tube was dissolved in 200 μl of PBS and was centrifuged for 2 min at 9000g, and the supernatant was discarded with a pipette. The pellet at the bottom of the tube was dissolved in 200 μl of PBS. 150 μl of the culture was taken to 96-well plate and its fluorescence intensity was measured by fluorimeter (FLA). The measured fluorescence intensity was corrected by subtracting the background fluorescence, measured in control wells containing 150 μl of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600). A strain, transformed with PtetR-GFP, which expresses GFP constitutively, was used as a positive control. A strain, transformed with Promoter less-GFP which does not express GFP was used as a negative control. | ||
Revision as of 22:11, 29 October 2008
| Main | Protcol | Parts Submitted to the Registry | Our Team | Acknowledgements |
|---|
Protocol
Reporter Assay
To quantitatively determine the performances of operator parts in the reporter plasmid, the change of fluorescence intensities of the reporter strains in the various concentrations of IPTG were measured. Overnight cultures of reporter strains grown at 37℃ in LB medium containing appropriate antibiotics were diluted 1:100 in LB medium and were incubated at 37℃ as fresh cultures. After their OD600 reached 0.6, the fresh cultures were diluted 1:1 in various concentrations of IPTG [0μM, 1 μM, 10 μM, 20 μM, 50μM, 100 μM and 1000 μM]. The fresh cultures were incubated at 37℃ for 12 hours and every 2 hours, 200 μl of each culture was taken to 1.5 ml tube and washed. Cultures were centrifuged for 2 min at 9000g, and the supernatant was discarded with a pipette. The pellet at the bottom of the tube was dissolved in 200 μl of PBS and was centrifuged for 2 min at 9000g, and the supernatant was discarded with a pipette. The pellet at the bottom of the tube was dissolved in 200 μl of PBS. 150 μl of the culture was taken to 96-well plate and its fluorescence intensity was measured by fluorimeter (FLA). The measured fluorescence intensity was corrected by subtracting the background fluorescence, measured in control wells containing 150 μl of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600). A strain, transformed with PtetR-GFP, which expresses GFP constitutively, was used as a positive control. A strain, transformed with Promoter less-GFP which does not express GFP was used as a negative control.
