Team:Tokyo Tech/Protocol

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(→Reporter Assay)
(→Reporter Assay)
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# Overnight cultures of reporter strains grown at 37℃ in LB medium containing appropriate antibiotics were diluted 1:100 in LB medium  
# Overnight cultures of reporter strains grown at 37℃ in LB medium containing appropriate antibiotics were diluted 1:100 in LB medium  
# Incubate at 37℃ as fresh cultures
# Incubate at 37℃ as fresh cultures
-
# After their OD<sub>600</sub> 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].  
+
# After their OD<sub>600</sub> 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].  
# The fresh cultures were incubated at 37℃ for 12 hours  
# The fresh cultures were incubated at 37℃ for 12 hours  
-
# Every 2 hours, 200 μl of each culture was taken to 1.5 ml tube and washed.  
+
# Every 2 hours, take 200 μl of each culture to 1.5 ml tube and '''wash''' it.  
-
## Centrifuge for 2 min at 9000g
+
-
##
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# Take 150 μl of the washed culture to 96-well plate and measure its fluorescence intensity by fluorimeter (FLA).
# Take 150 μl of the washed culture to 96-well plate and measure its fluorescence intensity by fluorimeter (FLA).
==== wash ====
==== wash ====
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Cultures were , 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.  
+
# Centrifuge for 2 min at 9000g
 +
# Discared the spernatant with a pipette
 +
# Dissolve the pellet at the bottom of the tube in 200 μl of PBS
 +
# Centrifuge for 2 min at 9000g
 +
# Discared the spernatant with a pipette
 +
# 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 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.
+
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 OD<sub>600</sub> and expressed in fluorescence per (ml x OD<sub>600</sub>). 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 22:51, 29 October 2008

Main Protcol Parts Submitted to the Registry Our Team Acknowledgements


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. Overnight cultures of reporter strains grown at 37℃ in LB medium containing appropriate antibiotics were diluted 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. The fresh cultures were incubated 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 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.

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