IGEM:IMPERIAL/2009/M3/Assays/IPTG effects

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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.