From 2009.igem.org

Thermoinduction Assay

Aims

To investigate the behaviour of the Harvard construct and show that when temperature is low (at 28 degrees), there is low fluorescence, while fluorescence increases when temperature is raised to 42 degrees.

Assay

Cells will be grown at 28 degrees. After equilibrating, they will be shifted to 42 degrees, and the change in fluorescence will be observed.

Equipment

Heated water bath
42 degrees incubator
Multi-well plate reader
96 well plates
15ml falcon tubes
50ml falcon tubes

Materials

M9 media with 0.2% Casamino acid supplemented with 1mM CaCl2 and 1mM MgSO4

Cells

Test construct CRP-GFP-Lac-RFP
Negative I13504 RBS-GFP-TT
Positive I13522 Ptet-RBS-GFP-TT

Protocol

Day 1 evening

1) Using a loop, pick out a single colony of the relevant cells from plates (in cold room)/
Or inoculate with starter culture 1 in 50.
Innoculate the cells on M9 + 0.5% Glucose and grow them overnight at 37 °C

2)Label the 15ml falcon tubes as following:

Tube 1: 28 degrees blank
Tube 2: 28 degrees Harvard-GFP construct
Tube 3: 28 degrees negative
Tube 4: 28 degrees positive
Tube 5: shift from 28 to 42 degrees blank
Tube 6: shift from 28 to 42 degrees Harvard-GFP construct
Tube 7: shift from 28 to 42 degrees negative
Tube 8: shift from 28 to 42 degrees positive
Tube 9: 42 degrees blank
Tube 10: 42 degrees Harvard-GFP construct
Tube 11: 42 degrees negative
Tube 12: 42 degrees positive

Label another set of tubes similarly.

3) Prepare 80ml of M9 media + 0.5% glucose from stock glucose solution
For 30% glucose stock, dilution factor = 30/0.5= 60x
Therefore, vol of glucose solution = (vol of M9 media)/60

Day 2

1) Calibrate the multi-well plate reader with the I13522 overnight cells

High gain= 50%
Low gain = 6% (no need to change)

2)The previously mentioned tubes are filled with 3ml of M9 + 0.5% glc

3)The overnight culture is diluted 1 in 20 into the first set of the newly prepared 15ml tubes

4) The tubes with cells are grown in the following conditions:

The 28 degrees cells and shift cells : at 28 degrees,
42 degrees cells : at 42 degrees

5)The water bath is switched on to 55 degrees

6) For the second set of tubes (without cells)

28 degrees tubes are put in the 28 degrees incubator
42 degrees tubes in the 42 degrees incubator
shift tubes (w/o cells) are placed in the 55 degrees water bath.

7) Subsequent OD and GFP readings are taken using the following settings:

Layout for the protocols can be imported as HVD-GFP
Measure: IPTG_GFP_FL (low gain)
Script: HVD-GFPhigain (high gain)

8) We need to ensure that bacterial cultures are at mid-log growth phase.

9) When mid-log phase is reached, an OD and GFP reading is taken of the cells. The tubes with cells are then added to the second set of tubes, and a subsequent OD and GFP reading is taken (time=0)

10) The second set of tubes (now with cells) are now grown at the following conditions:

28 degree tubes: at 28 degrees
Shift and 42 degree tubes: at 42 degrees

11) The cells are now sampled for their GFP and OD readings half hourly for around 5 hours until the GFP fluorescence plateaus off.

Team:Imperial College London/Wetlab/Protocols/Thermoinduction