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

Protocol

Day 1

Equipment

• 7ml sterile tubes x4
• 1.5ml Eppendorf tube x1
• Incubator 30^oC
• Gilson Pipettes

Reagents

• E.coli DH5α culture containing parts
• 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 30°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. Dilution series of 10μM (10^-5M) and 100μM (10^-4M)(This is to be diluted further by 10^-2 later)

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)

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

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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. 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 3-4 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

Under Construction (Do Not Edit!!)

After 4hrs in the shaker:

• Add a 200uL sample from each eppendorf tube to the 96 well plate.
• Do this for 8 repeats following suggested patterning (see above)
  • NOTE: Since all repeats are made from the same culture, it is enuogh to do 4 repeats - thus one plate can be used for 2 different tests. (Pipetting errors will be ruled out since absorbance is measured later and can then be considered when processing the data.)
• Add 4 x 200uL of growth medium to a well to act as a control.
• Take the plate and eppendorfs to BCHEM

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• Add 995ul of ultra pure water to the eppendorf, together with 5ul of undiluted GFP standard solution and mix __HIDER__

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A 200 x dilution of GFP is made.

The undiluted GFP is in the BCHM Level 6 Cold Room on our shelf. It is in a small grey plastic box. Pippettes, tips and ultrapure water are located on a shelf above the electroporation machine in the plate reader room.

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• Add 4 x 200uL of the 200x diluted GFP standard solution to the wells following the suggested patterning
• Take a reading
  • Take the plate to the plate reader room

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• Use the Victor3 to measure flourescence and absorbance __HIDER__

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Use the preprogrammed Assay under the 'Students' folder called GFP + Abs490

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• Repeat the measurment a further two times straight after each other __HIDER__

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This to assess the variability of the machine

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• Save data file from computer. __HIDER__

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You'll need a memory stick to save the information to. Unfortunatley the computer isn't networked

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• Copy and paste the data into a T9002/J37016/J37020 Data Spreadsheet

Equipment

• Incubator 30^oC
• Fluorometer
• Spectrometer
• Plate x1
• Plate Centrifuge
• 6x1.5ml tubes
• Gilson Pipettes
• Stop watch

Day 3:

Make 5 of 550 ml LB medium with 550 µl ampicillin in sterile conical flask Innoculate in 5 cultures into the the conical flasks Leave overnight at 37°C with shaking

Day 4:

Prepare a water bath at 30°C [should we use a shaking incubator?] Aliquot the 550 ml cultures into sterile tubes, 25 ml per tube Add 53.3075 ng AHL each time to increase [AHL] in steps of 10nM Begin the timer when AHL is added At 5 minute intervals for the first 40 minutes, and 10 minute intervals thereafter: Sample a 1 ml of the reaction mixture Immediately chill in ice (to slow the rate of reaction) Centrifuge at maximum speed for 30 seconds to pellet the cells Remove the supernatant and resuspend the pellet in 1ml distilled water Put in a spectrometer and excite it at 501nm and detect at 511nm [Note: Use solution at 0 minutes as a blank, assume no fluorescence is present prior to activation] These measurements should be repeated with the other 4 cultures of bacteria