IGEM:IMPERIAL/2008/Prototype/Wetlab/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

50mL flask (an additional one for each repeat)

Timer

Reagents
No antibiotic should be present in the media or plates unless the strain used has a natural resistance or a resistance gene has previously been integrated into the strain

LB Medium

LB Agar plates (approximately 18 per repeat)

Protocol
Be VERY careful to avoid contamination at all stages


 * 2 repeats should also be set up and the final data averaged or used together to obtain more acurate results


 * A trial run should be carried out to determine the approximate dilutions required for each time point to reduce the number of plates required


 * 1) Streak a culture of B.subtilis and allow to grow overnight
 * 2) Prepare 50mL of LB medium in a flask (ideally a 500mL or 1L flask), adding the required antibiotics (if any) and take 1mL to use as a blank
 * 3) Pipette 500μL into the 50mL of LB medium and mix thoroughly
 * 4) Immediately take 1mL of the new culture and measure the OD600 against the blank
 * 5) If the OD600 is above 0.8, dilute 10 fold and multiply the resulting absorbance by 10
 * 6) Carry out 10 fold dilutions (100μl of culture in 900μl of LB) until the culture should be approximately 10 fold more concentrated than the dilution at which the culture will form individual colonies (this should be determined by a trial run)
 * 7) Plate cells at the 10 fold higher dilution as well as the optimal diltuion and a 10 fold lower dilution (therefore 3 dilutions plated separately per time point)
 * 8) Be sure to mark the OD, time and dilution for each plate!
 * 9) Grow the B.subtilis at 37°C and 225rpm of mixing
 * 10) At 1 hour, 2 hours, 4 hours, 8 hours and overnight (~16 hours) take further 1 mL samples, measure OD, dilute and plate
 * 11) The following day, check all plates to determine which plate at each time point is the most crowded plate on which all the colonies can still be counted individually.
 * 12) The colonies on these plates should be counted on an illuminator
 * 13) To use the illuminator...
 * 14) The number of colonies on the plates should be multiplied by the dilution to obtain the colony forming units at each time point
 * 15) Plot a calibration curve of OD600 against cells per mL or OD600 against colony forming units