Team:Imperial College London/Wetlab/Protocols/IPTGgrowth

=IPTG effect on growth=

Aim

 * To produce a calibration curve to aid in the normalising of absorbance values. The relation of absorbance reading to number of cells varies with different cell strains. We are therefore doing one for Top-10.

Assay
Cultures of the E.coli with the relevant vector are grown to various cell densities. A sample of these cultures are taken and a dilution plate is carried out to work out approximate colony forming units per ml of culture. This data set is then combined with absorbance readings to create a graph relating the number of colony forming cells per ml to their absorbance measurements.

This curve then allows us to convert absorbance of a known volume of culture to colony forming units within the culture sample.

Aims

 * Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined
 * Determine the effect of IPTG toxicity on growth w/o any protein production complications

Assay
Normal cells (without any constructs) will be grown on M9 media until OD=0.7. IPTG of various concentrations will then be added, and the OD of the cells will be followed over time

Equipment

 * Multi-plate reader
 * 15ml falcon tubes
 * 96 well plates

Reagents
(for M9 minimal media preparation, refer to Secondary Carbon Sources)
 * M9 Minimal Media with 0.2% Casamino acids, 0.5% Glucose and Strep
 * LB Media

Things needed

 * LB + Strep

Inoculation of cells
1) Using a loop, pick out a single colony of Top-10 cells from a Top-10 Strep plate (in cold room). Innoculate the  cells on LB broth (with Strep)  and grow them overnight at 37 °C with spinning.

Day 2
1)	Before noon, check that the starter culture of Top-10 in LB has grown (ie. Turned cloudy). Take out and transfer to cold room

2)	At 3pm, dilute cells 1:50 (cells: media) into 10ml of supplemented M9 media. Grow them overnight at 37 °C

Things needed

 * Multi-well plate reader
 * 96 well plate
 * 1M IPTG solution (in freezer opp. side of microwave)
 * supplemented M9 minimal media
 * 15ml falcon tubes
 * Eppendorf tubes

Preparing IPTG stock solutions
1)	Take out an IPTG 1M aliquot from -20°C Freezer and wait to thaw

2)	Create 2 diluted solutions in 2 eppendorf tubes:

0.1M IPTG- add 100ul of 1M IPTG to 900ul of sterile H2O

0.01M IPTG- add 10ul of 1M IPTG to 1ml of sterile H2O

Mix well.

Growing up cells
1) Dilute the cells in fresh M9 media (5ml) 2)The OD is monitered by transferring 200ul aliquots into a 96 well plate and measuring the absorbance at 600nm.

IPTG experiment
Prepare a 96 well plate: 1) After the OD reaches 0.7, add in 200ul of Top-10 cells (from day 2) to wells A1 to A9.

2) Add the following volumes of IPTG to each of the labeled wells: A 1: 0 ul of IPTG ( 0 uM IPTG) A 2: 1 ul of 0.01M IPTG (50 uM IPTG ) A 3: 2ul of 0.01M IPTG (100 uM IPTG) A 4: 0.5ul of 0.1M IPTG (250 uM IPTG) A 5: 1.0ul of 0.1M IPTG (500 uM IPTG) A 6: 1.5ul of 0.1M IPTG (750 uM IPTG) A 7: 2.0ul of 0.1M IPTG (1.0 mM IPTG) A 8: 0.5ul of 1M IPTG (2.5 mM IPTG) A 9: 1.0ul of 1M IPTG (5.0 mM IPTG) A10: Blank Perform 2 replicates Ie. A1 to A10 Up till C10 Mix well. 3) To blank well, add in 200ul of M9+ 0.5% Glucose 4) Set up the plate reader to take OD readings at 600nm every half hour. (Using script IGEM Abs). Obtain readings overnight. Note 1: Do a trial run sometime during the day to ensure that the script runs properly (if not your overnight measurements might not give any results!) Note 2: Readings after 8 hours might not longer be accurate due to evaporation. 5)Determine background absorbance by measuring control well. This should be subtracted from subsequent absorbance readings.