Team:Imperial College/Growth

Aim

To produce a calibration curve to aid in the normalising of fluorescence values to allow proper characterisation of Promoters and RBSs for B.subitlis. This protocol must give results that are as accurate as possible over a considerable range of Optical Densities.

Equipment

• Spectrophotometer
• Cuvettes
• P20, P200 and P1000 Gilsons

Reagents

• 20ml of LB in a 250ml flask
• B. subtilis transformed with antibiotic selectable marker
• LB agar plates with antibiotic

Protocol

1. Inoculate 20ml of LB broth with B. subtilis containing a selectable marker, we used B. subtilis transformed with spectinomycin. Incubate overnight at 37^oC.
2. Collect culture and measure the O.D.₆₀₀.
3. With the O.D.₆₀₀ calculate how much of the overnight culture is required to give an O.D.₆₀₀ of 0.5, 1, 1.5, 2, 2.5, 3 in 2ml using the following calculation:
   • Vol of culture (ml)=(O.D.₆₀₀ wanted / O.D.₆₀₀ of Overnight culture) * 2ml
4. Carry out the dilutions required using LB media to make the volumes up to 2ml.
5. Remove 1ml of each dilution and then measure the O.D.₆₀₀ and discard.
6. Now carry out a series of 10 fold dilutions by removing 100ul of the dilution and adding into to 900ul of LB (this gives 10^-1 dilution) carry this on until a suitable dilution is reached. We used the following dilutions:
   • O.D₆₀₀ of 0.5 = x10^-6 and x10^-7
   • O.D₆₀₀ of 1 = x10^-6 and x10^-7
   • O.D₆₀₀ of 1.5 = x10^-6 and x10^-7
   • O.D₆₀₀ of 2 = x10^-6 and x10^-7
   • O.D₆₀₀ of 2.5 = x10^-6 and x10^-7
   • O.D₆₀₀ of 3 = x10^-7 and x10^-8
7. Plate 100ul of the appropriate dilutions onto an LB agar plate containing the antibiotic selectable marker.
8. Grow the plates at 37°C overnight
9. The following day count the number of colonies on the plates and multiply up by the dilution to obtain the colony forming units at each time point. Remember when plating the cells only 100ul of 1000ul was used to plate and so you need to multiply by a further 10.