IGEM:IMPERIAL/2009/M0/Assays/Phase1+

General Phase 1

Aims

• To tie in the different growth curves of normal conditions and protein production conditions

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.

Equipment

• Spectrophotometer

Reagents

• M9 medium with kanamycin(20 ug/ml)

Protocol

1. 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 15g/L glucose 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 with 10g/L.

5. A 500 ul aliquot was taken out and put in a cuvette.

6. OD Absorbance readings were measured on a spectrophotometer at 600nm.

7. The OD measurements were repeated until OD is around 0.65 (around 6 hours).

8. The glucose concentration was determined using the glucose assay kit.

9. After the glucose concentration is determined (takes slightly more than half an hour), this is correlated to the previously created standard curve (see growth modelling). Therefore, the current residual glucose concentration can be estimated.

10. IPTG is added, and glucose is topped up to 10g/L. (or other concentration based on Phase 1-2 results) This kickstarts the protein production, and the growth model is changed to that of the protein production phase. The timer using glucose concentration also starts ticking