IGEM:IMPERIAL/2009/M3/Assays/IPTG effects2

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 will then be added, and the OD of the cells will be followed over time.

Equipment

• Spectrophotometer
• 600nm absorbance filter
• 15ml falcon tubes
• cuvettes

Reagents

Media

Total volume: 120ml

M9 minimal media – 11.28g/L of 5x powder
MgSO4 – 0.4g/L
Glycerol (5.0g per L)= 54.39uM 0.5%
Glucose (0.5g per L) = 2.78mM 0.05% per 100ml
kanamycin (20 ug/ml) = 1mg/500ml

Cultures

E.coli cells (TOP-10 strain)

Others

IPTG (0.1 g)

Protocol

Thurs 11AM:

Things needed

• Minimal Media constituents

M9 Minimal Media Preparation:

• Measure out the following reagents and dissolve them in 1000ml of sterile H20:

M9 Minimal Media- 11.28g/L of 5x powder
Glucose (0.5g per L) = 2.78mM 0.05%
Glycerol (5.0g per L)= 54.39uM 0.5%
Maltose (5.0g per L)= 14.6mM 0.5% per 100ml

Autoclave the M9 media to ensure sterility.

MgSO4 - 0.4g/L powder
To maintain sterility, MgSO4 solution should be filter sterilised (Minisart® 0.20µm syringe filter) and added after the other chemicals had been dissolved, mixed and autoclaved

Thurs 1PM:

Things needed

• Minimal Media constituents
• 15ml falcon tubes

Inoculation of cells

• Inoculate single colonies of E. coli cells into 15ml falcon tubes containing 5 ml of the pre-warmed (37°C) supplemented M9 medium with spectinomycin (20 ug/ml)

• Grow the cultures for O/N with spinning at 70 rpm.

Will take 20hrs based on TOP10-DH5a in Jason Kelley Paper

Fri 9AM:

Things needed

• IPTG 0.1g
• Minimal Media constituents
• 15ml falcon tubes

1) Growing up of cultures

• Dilute the cultures which are at a high cell density 1:20 into 2 conical flasks of 50 ml of fresh media each and grow the cultures at 28°C in an incubator.
  

2) IPTG solution Preparation:

• 0.1g of IPTG dissolved in 0.4ml of dH2O (filter sterilize) to get 1M IPTG

3) Labelling of plates

• Label 2 conical flasks setups:
  

Flask 1: 0 uM IPTG
Flask 2: 1 mM IPTG

Fri 12PM:

Things needed

• 1M IPTG solution
• Spectrophotometer
• Cuvettes

Monitering of OD

• After 3 hours, 1ml of solution from each flask is transferred to a cuvette.

• The OD is obtained by measuring the absorbance at 600nm using the spectrophotometer. The OD should be around 0.7. If not, wait longer.

• After the OD reaches 0.7, add 49ul of 1M IPTG to flask 2 and mix well.

• Repeat the absorbance measurement every hour for both flasks during mid-exponential growth for 6 hours

• Determine background absorbance by measuring blank with only media. This should be subtracted from subsequent absorbance readings.