IGEM:IMPERIAL/2009/M3/Assays/tempshift

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Contents

Effect of Temperature

Aims

We aim to investigate the behaviour of the pLambda promoter when subjected to a change in temperature.

Assay

For this assay, we will measure the response of pLamda promoter to increasing temperatures. We want to monitor the promoter activity to plot a graph of activity against temperature. We will fit this to a hill function to model our pLambda promoter. This reates to M3 since we will be using an increase in temperature to induce M3 and result in the beginning of cell death.

Equipment

1) Thermometer

2) Heated water bath

Reagents

1) tetracycline, 10 μg ml−1

2) ampicillin, 100 μg ml−1

3) 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (XGal), 0.04 mg ml−1.

4) LB broth

Protocol

1) Mix:

  • tetracycline, 50 μg
  • ampicillin, 500 μg
  • 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (XGal), 0.20 mg
  • 5mL of LB (Luria-broth)

and plate e. coli. Grow overnight at 28 °C.


2) We need to ensure that bacterial cultures are at mid-log growth phase, by measuring the optical density at 600 nm (OD600).

3) Check if cell count shows that bacterial is growing at mid-log phase.

4) If mid-log phase is reached, transfer cultures to water baths at 28 °C, 30°C, 36°C, 39°C, 42°C, respectively, with both a control and a transformed culture

5) OD600 and GFP/beta-galactosidase readings (refer to Assays) should be taken at half hourly intervals.

Wetlab processing

A graph of temperature against cell count would be plotted to access the viability of e. coli with regards to temperature.

References

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