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

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m (Inoculation of cells)
Current revision (16:07, 26 August 2009) (view source)
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* Minimal Media constituents
* Minimal Media constituents
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* Prepare spectinomycin alliquots
==== M9 Minimal Media Preparation:====
==== M9 Minimal Media Preparation:====

Current revision

Contents

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
  • Prepare spectinomycin alliquots

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.
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