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)

Preparation of diluted GFP standard solution

• 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 (IS THIS POSSIBLE??!?!)

1. Add 2µl of stock concentrations of AHL (10μM & 100μM) to the respective wells as shown.

2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone (3OC6HSL), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of 3OC6HSL. Three wells were each filled with 200 µl of medium to measure the absorbance background

Under Construction (Do Not Edit!!)

• To start AHL incubation:
  • Label each tube with AHL concentration
  • Put 20ul of the AHL into 11 seperate 5ml white capped tubes
  • Add appropriate amount of T9002 samples as per the table below
  • Record time of inoculation in report sheet.
  • Vortex each tube

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• Incubate all 5mL tubes in a 37°C shaker for 4 hours so GFP expression can reach steady state __HIDER__

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Do not pipette the samples into the 96 well plate yet - because if the 96 well plate is put in the shaker, cross-contamination between the well is very likely to happen.

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Loading Plate

1. Follow the schematic for the plate adding everything but the DNA sample.
2. Place the top on the plate and place in the incubator. Leave for a few minutes to heat to 30^oC
3. Remove from incubator and centrifuge for 1 minute
4. Remove lid and Measure in the flourometer.
5. Then to begin the reaction add .... purified DNA sample.
6. Place lid back on and place back in the incubator at 30^oC

Schematic

Well Test Construct Stock Concentration AHL (ul) Final AHL Concentration A1 pTet Commercial E.coli extract A2 pTet Commercial E.coli extract A3 pTet Commercial E.coli extract A4 pTet Our S30 Cell extract A5 pTet Our S30 Cell extract A6 pTet Our S30 Cell extract A7 None Commercial E.coli extract A8 None Our S30 Cell extract B1 pT7 Commercial T7 extract B2 pT7 Commercial T7 extract B3 pT7 Commercial T7 extract B4 pT7 Our S30 Cell extract B5 pT7 Our S30 Cell extract B6 pT7 Our S30 Cell extract B7 None Commercial T7 extract B8 None Our S30 Cell extract

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