Team:Imperial College London/Wetlab/Protocols/IPTG-RFP

=Lac characterisation and IPTG effect on protein production=

Aims

 * Characterise Lac promoter by varying amounts of IPTG
 * Determine the IPTG concentration that allows for maximal protein production while still being non-toxic to the cell

Assay
The cells will be grown until OD=0.7. Now, IPTG of various concentrations will be added, and the RFP output will be measured. The experiment will generate OD and fluoresence data for RFP The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection for CRP promoter) The glucose concentration will be taken from commercial autoinduction media (0.05%) – takes about 7 hours to exhaust

Equipment

 * Plate reader
 * 96 well plates
 * 15ml falcon tubes

Reagents

 * Supplemented M9 Minimal Media with 0.5% secondary carbon source and 0.05% glucose (with Strep)
 * LB Media (with Strep)
 * 1M IPTG stock solution

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 stock solution
 * supplemented M9 minimal media
 * 15ml falcon 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: 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

Growing up cells
1) Dilute the cells 1:20 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. (The IPTG values will vary depending on results of IPTG-growth) 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 and RFP readings at 600nm every half hour. (Using script IGEM Fluor Abs but changing measurement protocol to RFP iGEM). 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. Acorrected = A - Ablank

6) Determine background fluorescence at different ODs from the fluorescence of control cells without a GFP expressing vector. (This experiment has already been done- see IPTG effects on growth) Fluor corrected = Fluor - Fluor cells

7. The data was then converted to absolute units (CFU/well and GFP molecules/well) using the calibration curves described in the calibration assays.

8. The GFP synthesis rate, S, can be described below: Stotal =dGFP/dt Scell = Stotal/CFU

9. From modelling, we can calculate PoPS(t)



where PoPS(t) is the time dependent rate of mRNA synthesis Scell is the GFP synthesis rate per cell (GFP over time / Cell no) gammaI is the degradation rate of immature GFP (incorporating degradation and dilution due to cell growth) gammaM is the degradation rate of mRNA a is the maturation rate of GFP rho is the constant rate of protein synthesis per mRNA