IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.2

Protocol(Status: ???)

• define status: Work in Progress / Feedbacks Required / Validated (other tags ?)

Day 1

Equipment

• 7ml sterile tubes x4
• 1.5ml Eppendorf tube x1
• Incubator 37^oC
• Gilson Pipettes

Reagents

• E.coli BL21; culture containing pTet-LuxR-pLux-GFP
• LB medium

• Ampicillin stock (50 mg/ml)
• GFP Standard Solution
• AHL stock

Innoculation of Media

1. Inoculate 10ul of stored parts 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)

Preparation of diluted series of AHL
http://parts.mit.edu/registry/images/4/4c/F2620-TF-Small.png
(Taken off BBa_F2620. Results show required dilution of AHL solution)

1. Dilute the powder AHL into a stock solution for 1mM. 50ml of it will be made. The stock solution will then be diluted 10x, of which 0.6ul will be added to 60ul of the cell extract.

Preparation of diluted GFP standard solution

1. Add 995ul of ultra pure water an eppendorf tube, together with 5ul of undiluted GFP standard solution and mix. (This gives a 200x dilution to be used as a positive control)

Day 2

Equipment

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

Reagents

• LB medium
• Ampicillin stock (50 mg/ml)
• Diluted GFP standard solution
• 10μM AHL stock
• 100μM AHL stock

Innoculation of Media (cont'd)

1. In the morning, prewarm 20 mL LB Amp medium in the 30°C water bath.
2. Measure and record OD600
3. Inoculate a 10ml fresh culture from the o/n to bring back the OD600 to 0.1, use the prewarmed LB + Ampicilin in waterbath. (Using prewarmed LB prevents a temperature shock to the culture, which would increase lag time)

<amsmath>

\frac{0.1}{\mbox{OD of culture}} \times \mbox{10 mL}

</amsmath>

OD of XXX culture (1st Measurement): _________

Amount to dilute of XXX culture = ________ mL (amount of original culture to use)

Amount of prewarmed LB with Ampicillin to use = ________ mL (16 mL - above result)

1. This returns cells to exponential phase from stationary phase
2. Return LB to 30°C waterbath.

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??!?!)

Standard GFP solution added as a positive control.

1. Add 2µl of stock concentrations of AHL (10μM & 100μM) to the respective wells as shown.
2. Place the top on the plate and centrifuge for 1 minute
3. 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

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.
2. Repeat measurements after every 5 minutes until the fluorescence is constant
3. Before every measurement in the fluorometer spin the plates in a plate centrifuge
4. Save data file from computer
5. Copy and paste the data into an Excel spreadsheet for data analysis