Team:Osaka/Protocols

From 2011.igem.org

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(→Cell survival assay 2: Mitomycin C)
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#Pipette 50μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.  
#Pipette 50μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.  
#Wrap plates in aluminium foil and incubate at 37°C.
#Wrap plates in aluminium foil and incubate at 37°C.
-
#After 16h, count number of colonies formed on control (non-mitomycin C) and UV-irradiated plates.
+
#After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.
-
=== SOS Promoter assay ===
+
 
 +
=== SOS promoter assay 1: Carotenoid biosynthesis ===
#Pre-culture transformed cells in 8ml of LB medium at 37°C for 12h.
#Pre-culture transformed cells in 8ml of LB medium at 37°C for 12h.
#Transfer pre-culture to OD600 measurement dish (ø50mm).
#Transfer pre-culture to OD600 measurement dish (ø50mm).
#Irradiate with UV light at desired energy dosage.
#Irradiate with UV light at desired energy dosage.
#Incubate irradiated cells for a further 2h.
#Incubate irradiated cells for a further 2h.
-
#Measure OD600 as a measure of cell density.
+
#Measure OD600 as a surrogate for cell density.
#Transfer cells into 15ml Falcon tubes and centrifuge.
#Transfer cells into 15ml Falcon tubes and centrifuge.
#Discard supernatant, add 1ml water to wash cells.
#Discard supernatant, add 1ml water to wash cells.
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#Add 500μl acetone and vortex.
#Add 500μl acetone and vortex.
#Incubate at 55°C for 15min.
#Incubate at 55°C for 15min.
-
#Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 measurements.
+
#Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 values.
 +
 
 +
 
 +
=== SOS promoter assay 2: GFP ===
 +
#Pre-culture transformed cells in 8ml of LB medium at 37°C for 12h.
 +
#Transfer pre-culture to OD600 measurement dish (ø50mm).
 +
#Irradiate with UV light at desired energy dosage.
 +
#Incubate irradiated cells for a further 2h.
 +
#Measure OD600 as a surrogate for cell density.
 +
#Transfer cells into 15ml Falcon tubes and centrifuge.
 +
#Discard supernatant, add 1ml water to wash cells.
 +
#Repeat centrifugation and decantation.
 +
#Resuspend cells in 1ml water.
 +
#Measure fluorescence at 395nm excitation and 509nm emission. Use pure water as blanks, and divide emission values by OD600 values.
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Revision as of 11:12, 28 October 2011

Contents

Protocols

Cell survival assay 1: UV irradiation

  1. Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
  2. Induce parts with IPTG addition (to final concentration of 100µM) for 1h.
  3. Dilute pre-culture samples according to intended UV energy dosage (if dosage is high, lower dilution rate is needed as fewer cells are expected to survive).
  4. Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
  5. Irradiate cells on the agar with UV light at desired energy dosage.
  6. Wrap plates in aluminium foil and incubate at 37°C.
  7. After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.


Cell survival assay 2: Mitomycin C

  1. Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
  2. Induce parts with IPTG addition to final concentration of 100µM and incubate for 1h.
  3. Add mitomycin C to desired final concentration (we used 2µg/ml) and incubate at 37°C for a further 2h.
  4. Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.
  5. [RECOMMENDED] Dilute pre-culture samples according to expected survival rate, as determined from a preliminary experiment (lower dilution rate is needed if fewer cells are expected to survive).
  6. Pipette 50μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
  7. Wrap plates in aluminium foil and incubate at 37°C.
  8. After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.


SOS promoter assay 1: Carotenoid biosynthesis

  1. Pre-culture transformed cells in 8ml of LB medium at 37°C for 12h.
  2. Transfer pre-culture to OD600 measurement dish (ø50mm).
  3. Irradiate with UV light at desired energy dosage.
  4. Incubate irradiated cells for a further 2h.
  5. Measure OD600 as a surrogate for cell density.
  6. Transfer cells into 15ml Falcon tubes and centrifuge.
  7. Discard supernatant, add 1ml water to wash cells.
  8. Repeat centrifugation and decantation.
  9. Add 500μl acetone and vortex.
  10. Incubate at 55°C for 15min.
  11. Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 values.


SOS promoter assay 2: GFP

  1. Pre-culture transformed cells in 8ml of LB medium at 37°C for 12h.
  2. Transfer pre-culture to OD600 measurement dish (ø50mm).
  3. Irradiate with UV light at desired energy dosage.
  4. Incubate irradiated cells for a further 2h.
  5. Measure OD600 as a surrogate for cell density.
  6. Transfer cells into 15ml Falcon tubes and centrifuge.
  7. Discard supernatant, add 1ml water to wash cells.
  8. Repeat centrifugation and decantation.
  9. Resuspend cells in 1ml water.
  10. Measure fluorescence at 395nm excitation and 509nm emission. Use pure water as blanks, and divide emission values by OD600 values.
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