IGEM:IMPERIAL/2009/M3/Assays/IPTG effects
Dry lab test experiment: IPTG effect on growth
Aims
- Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined
- Determine the effect of IPTG toxicity on growth w/o any protein production complications
Assay
Normal cells (without any constructs) will be grown on M9 media supplemented with glucose and secondary carbon source until OD=0.7
IPTG of various concentrations will then be added, and the OD of the cells will be followed over time
Updated version
Equipment
- Spectrophotometer
- 600nm absorbance filter
- 17mm tubes
- 96 well plates
Reagents
Media
M9 Minimal Media
Disodium Phosphate (12.0g)
Potassium dihydrogen phosphate (6.0g)
Sodium Chloride (1.0g)
Ammonium Chloride (2.0g)
Magnesium Sulphate (0.75g)
Glycerol (5.0g per L)= 54.39uM 0.5%
Glucose (0.5g per L) = 2.78mM 0.05% per 100ml
Others
IPTG (2 g)
Protocol
Day 1 4PM:
Omit this day if there are already available cells in culture and do the minimal media preparation in day 2
Things needed
- Minimal Media constituents
- 17mm tubes
1) M9 Minimal Media Preparation:
- Measure out the following reagents and dissolve them in 1000ml of sterile H20:
Disodium Phosphate = 6.0g
Potassium dihydrogen phosphate = 3.0g
Sodium Chloride = 0.5g
Ammonium Chloride = 1.0g
Glycerol (5.0g per L)= 54.39uM
Glucose (0.5g per L) = 2.78mM
2) Inoculation of cells
- Inoculate single colonies of E. coli cells into 17mm tubes containing 5 ml of the pre-warmed (37°C) normal supplemented M9 medium with kanamycin (20 ug/ml)
- Grow the cultures for O/N with spinning at 70 rpm.
Day 2 4PM:
Start from here if there are already available cells in culture
Things needed
- IPTG
- Minimal Media constituents
- 17mm tubes
1) IPTG solution Preparation:
- 1g of IPTG dissolved in 4ml of dH2O (filter sterilize) to get 1M IPTG
- 100ul of 1M IPTG further diluted in 1ml of culture to give 0.1M IPTG
2) Growing up of cultures
- Dilute the cultures which are at a high cell density 1:1000 into 5 ml of fresh media in a 17mm tube and grow the cultures at 28°C in 28°C incubator O/N
3) Labelling of plates
- Label 96 well plate setups:
Well 1: control: no cells
Well 2: 0 uM IPTG
Well 3: 50 uM IPTG
Well 4: 100 uM IPTG
Well 5: 250 uM IPTG
Well 6: 500 uM IPTG
Well 7: 1.0 mM IPTG
Well 8: 2.5 mM IPTG
Well 9: 5.0 mM IPTG
Day 3 9AM:
Things needed
- Spectrophotometer
- 600nm absorbance filter
Monitering of OD
- Add the following volumes of IPTG to each of the labeled wells:
Well 1: 0 uM IPTG ---0 ul of IPTG
Well 2: 0 uM IPTG ---0 ul of IPTG
Well 3: 50 uM IPTG ---0.1 ul of 0.1M IPTG
Well 4: 100 uM IPTG---0.2ul of 0.1M IPTG
Well 5: 250 uM IPTG---0.5ul of 0.1M IPTG
Well 6: 500 uM IPTG---1.0ul of 0.1M IPTG
Well 7: 1.0 mM IPTG---2.0ul of 0.1M IPTG
Well 8: 2.5 mM IPTG---0.5ul of 1M IPTG
Well 9: 5.0 mM IPTG---1.0ul of 1M IPTG
- The OD is monitered by transferring 200ul aliquots into a 96 well plate and measuring the absorbance at 600nm.
- After the OD reaches 0.7 (should be immediately), transfer 200 ul aliquots from each culture into the labelled flat-bottomed 96 well plate with IPTG (all except well 1)and mix well.
- Incubate the plate in a multi-well spectrophotometer at 28°C (water bath?) and assay with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid) and shaking (3 mm, linear, normal speed, 15 seconds)
- Repeat the absorbance measurement every hour during mid-exponential growth for 6 hours
- Determine background absorbance by measuring control well. This should be subtracted from subsequent absorbance readings.