Team:Tokyo Tech/Protocol

From 2008.igem.org

Protocol

Reporter Assay

figure Wash

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.

1. Dilute overnight cultures of reporter strains grown at 37℃ in LB medium containing appropriate antibiotics 1:100 in LB medium
2. Incubate at 37℃ as fresh cultures
3. After their OD₆₀₀ reach 0.6, dilute the fresh cultures 1:1 in various concentrations of IPTG [0μM, 1 μM, 10 μM, 20 μM, 50μM, 100 μM and 1000 μM].
4. Incubate the fresh cultures at 37℃ for 12 hours
5. Every 2 hours, take 200 μl of each culture to 1.5 ml tube and wash it.
6. Take 150 μl of the washed culture to 96-well plate and measure its fluorescence intensity by fluorimeter (FLA).

Wash

1. Centrifuge for 2 min at 9000g
2. Discared the spernatant with a pipette
3. Dissolve the pellet at the bottom of the tube in 200 μl of PBS
4. Centrifuge for 2 min at 9000g
5. Discared the spernatant with a pipette
6. Dissolve the pellet at the bottom of the tube in 200 μl of PBS

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 P_tetR-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.

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figure 3-4.Protocol for apply pressure to a display.

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