Team:Imperial College London/Wetlab/Protocols/SecondaryCarbon

=Assay for Diauxie cell growth and Secondary carbon source selection=

Aims

 * To generate the diauxie growth curves, as well as normal growth curves for Top-10 for modelling
 * To determine the best secondary carbon source for optimal growth

Assay
Cells are allowed to grow at 28°C, and their growth ie OD would be monitored using a plate reader, at 600nm, at regular intervals overnight. A plot of OD vs time will be generated. This result is to be fitted to a diauxie growth model. A control with only glucose and no secondary carbon source is used. This control will give us normal growth curves of Top-10 cells.

Materials
1)	M9 Minimal Media (without glucose) (makes 1000mL) 1.	M9 minimal powder 11.28g 2.	Casamino acid 2g 3.	Magnesium Sulphate powder (to be made into filter sterilised 1M MgSO4) 4.	Calcium chloride powder (to be made into filter sterilised 0.1M CaCl2) 5.	~2000mL of sterile H2O 2)	Carbon source (filter sterilised) a.	2g Glucose powder (to be made into filter sterilised 20% Glucose solution ie. 20g in 100ml) b.	2g each secondary carbon source (to be made into filter sterilised 10% of each secondary carbon source ie. 10g in 100ml) 3)	Streptomycin (1 : 1000 dilution) Equipment: 1)	15ml falcon tubes 2)	50ml falcon tubes 3)	Cuvette 4)     Spectrometer 5)	LB broth (with Strep) 6)	2000mL conical flask 7)	Multi-Plate reader 8)     96 well plate

Day 1
1) Prepare M9 minimal media by carefully weighing out the required chemicals except MgSO4, and dissolve them in 1000mL of sterile H2O. 1.	Weigh out 11.28g of M9 minimal powder and 2g of casamino acid in 1000ml of sterile H2O 2.	Send for autoclave (ie before 12.30pm) 3.	Dissolve MgSO4 as per formal weight to form 1M MgSO4. Dissolve CaCl2 as per formal weight to form 0.1M CaCl2 4.	Filter sterilise into the M9 media using Minisart® 0.20µm syringe filter 5.	Add 1mL of antibiotic strep to the media. 6.	If no carbon source specified, make up 0.5% Glucose.  If carbon source specified, make up 0.5% of secondary carbon source and 0.05% Glucose. 2)	Prepare secondary carbon source stock solution 1.	Dissolve carbon source in 10ml sterile H2O to form 10 or 20% stock solutions as per above. (using idea that 1% = 1g in 100ml H2O). Important note: Add half the sterile H2O first, then add in carbon source powder slowly while vortexing. Finally add in all the sterile H2O. 2.	Filter sterilise into new 15ml falcon tubes using Minisart® 0.20µm syringe filter. Label new tubes as filtered stock solutions. 3)	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

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

Day 3
1) Prepare 6 x 50ml falcon tubes and 1x eppendorf tube. Tube 1: M9 + 0.5% glucose (control) Tube 2: M9 + 0.5% arabinose + 0.05% glucose Tube 3: M9 + 0.5% xylose + 0.05% glucose Tube 4: M9 + 0.5% rhamnose + 0.05% glucose Tube 5: M9 + 0.5% galactose + 0.05% glucose Tube 6: M9 + 0.5% maltose + 0.05% glucose Eppendorf tube: blank of secondary carbon sources.  (M9 + any sugar. Sugars do not affect OD much) 2) To each 50ml tube, pipette in 40ml of M9 media 3) To each 50ml tube, add in the carbon source stock solutions to make up 0.5% secondary carbon source. Mix well. 4) Add 1ml of starter culture Top-10 cells to each 50ml tube (1:40 dilution). Mix well 5) Pipette out 1ml from each 50ml tube into a cuvette. These will be read using the spectrometer, with the eppendorf contents serving as a blank.  The blank can be reused. 6) Place in 28°C incubator with shaking. 7) Take similar spectrometer readings hourly. From the 5th or 6th hour. the graph should start to plateau off.  Take half hour readings from 5th hour to more accurately determine shape of graph. 8) During the 5th hour ,pipette out 200uL from each 50ml tube  and put it in well A1 to A6 of the 96 well plate. A7 is the blank well. Perform 2 replicates for each tube Now, wells A1-C6 will be filled with samples 9) Set up the plate reader to take OD readings at 600nm at specified intervals. (Using script IGEM Abs). Obtain readings overnight. Note: readings after 8 hours might not longer be accurate due to evaporation. 10) Determine background absorbance by measuring control well. This should be subtracted from subsequent absorbance readings.