IGEM:IMPERIAL/2009/M0/Assays/Growth Curve

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Contents

Growth Curve Modelling

Aims

  • To determine the glucose utilisation rate as well as cell growth rate with time.
  • By using the glucose assay kit and OD measurements, a quantitative measurement of glucose concentration as well as cell density can be measured.

Assay

E.coli cells are grown in conditions where glucose is not limiting. A standard amount of inoculum is added to a standard starting concentration of glucose.

The drop in glucose concentration is monitered by the glucose assay kit, while the growth of cells is monitered by taking OD readings.

This is monitered at regular time intervals to generate a growth curve at 28°C.

Equipment

  • Spectrophotometer

Reagents

  • M9 medium with 15g/l glucose

Protocol

1. 3 setups were created:
a) cells with promoter constructs
b) cells with promoter constructs and IPTG (protein production induced)
c) control cells

2. Single colonies of E. coli cells were inoculated into 17 mm test tubes containing 5 ml of pre-warmed (28°C) supplemented M9 medium with kanamycin (20 ug/ml) corresponding to the above 3 setups.

3. Cultures were grown for approximately 12 hrs at 28°C with spinning at 70 rpm.

4. We then diluted the cultures 1:100 into 100 ml of pre-warmed fresh media. (conc?)

5. We measured the OD600 of a 500 ul aliquot in a cuvette from each culture on a spectrophotometer. The same aliquot is used with the glucose assay kit to determine the concentration of glucose.

6. The OD and glucose concentration measurements were repeated every half an hour until the OD starts to flatten out.

7. A graph of OD and glucose concentration can be plotted

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