IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.1

Protocol

Day 1

Equipment

• 7ml sterile tubes x4
• 1.5ml Eppendorf tube x1
• Room temperature 25^oC
• Gilson Pipettes

Reagents

• E.coli BL21; culture containing parts :pTet-GFP, pT7-GFP, pcI-GFP
• LB medium

• Ampicillin stock (50 mg/ml)
• GFP Standard Solution (what is the stock concentration ?)

Innoculation of Media

1. Inoculate 10ul of transformed E.coli cells in individual 2ml LB medium containing 2ul of ampicillin
2. Incubate at 37°C for overnight in a shaker. (This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase)

Day 2

Equipment

• Water bath @ 30°C
• Spectrophotometer
• Stripettes
• Well-plate x1
• Plate Centrifuge
• Stop watch

(Tubes ? volume ? quantity ?)

Reagents

• LB medium
• Ampicillin stock (50 mg/ml)
• Diluted GFP standard solution

Loading Plate

1. Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. (Follow the schematic as shown)

Three wells to be filled with 200µl of media to measure the absorbance background.

Three further wells to be filled with 200 µl of (media + empty-vector culture) to measure fluorescent background. (IS THIS POSSIBLE??!?!) (Which empty vector ? it should have the same antibiotic and the cells should also come from an overnight culture)

Standard GFP solution added as a positive control.

1. Place the top on the plate and centrifuge for 1 minute (Centrifuge ? or shacker ? if centrifuge, at which speed ?)

1. Remove lid and measure in the flourometer.

(Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units)

1. Repeat the measurement a further two times straight after each other (This is to test the variability of the machine)
2. Place lid back on and place back in the incubator at 30^oC

(Are you not tracking the growth of the culture too with the absorbance ? How can you normalise your fluorescence then ?) (Are you continuously sampling from the 10ml culture ? or are you re-using the cells in the 96-well plate ?)

Schematic

Well Test Construct Stock Volume (ul) AHL (ul) Final [AHL] A1 pTet-GFP 200 0 0 A2 pTet-GFP 200 0 0 A3 pTet-GFP 200 0 0 A5 LB-Amp Media 200 0 0 A6 LB-Amp Media 200 0 0 A7 LB-Amp Media 200 0 0 B1 pT7-GFP 200 0 0 B2 pT7-GFP 200 0 0 B3 pT7-GFP 200 0 0 B5 LB-Amp Media + Non-expressing culture 200 0 0 B6 LB-Amp Media + Non-expressing culture 200 0 0 B7 LB-Amp Media + Non-expressing culture 200 0 0 C1 pcI-GFP 200 0 0 C2 pcI-GFP 200 0 0 C3 pcI-GFP 200 0 0 C5 Diluted GFP Solution 200 0 0 C6 Diluted GFP Solution 200 0 0 C7 Diluted GFP Solution 200 2 0 D1 pTet-LuxR-pLux-GFP + 10μM AHL 200 2 10-7M D2 pTet-LuxR-pLux-GFP + 10μM AHL 200 2 10-7M D3 pTet-LuxR-pLux-GFP + 10μM AHL 200 2 10-7M E1 pTet-LuxR-pLux-GFP + 100μM AHL 200 2 10-6M E2 pTet-LuxR-pLux-GFP + 100μM AHL 200 2 10-6M E3 pTet-LuxR-pLux-GFP + 100μM AHL 200 2 10-6M

Collecting Data

1. After 5 minutes of incubation measure the fluorescence by repeating procedure 3-5 above. (By doing so many measurements, aren't you worried to disrupt the growth of the culture with an ever changing temperature ?)

1. Repeat measurements after every 5 minutes until the fluorescence is constant (Cells are growing, so the fluorescence of the culture will not be constant before they reach stationary? Are you looking for single cell fluorescence steady-state ?)

(You mentioned that you use only 1 96-well plate ? How do you deal with multiple measurements ?)

1. Before every measurement in the fluorometer spin the plates in a plate centrifuge
2. Save data file from computer
3. Copy and paste the data into an Excel spreadsheet for data analysis