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

Equipment

 * Spectrophotometer
 * 600nm absorbance filter
 * 17mm tubes
 * 96 well plates

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)

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

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.