Initial Testing of DNA Construct In Vivo

Aims

• To test to see if the DNA construct from the registry are viable. This is done in vivo.
• The constructs are pTet-LuxR-pLux-GFP.

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)
• 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 culture for AHL induction and GFP measurement From the stock culture of transformed E.coli, dilute the culture so that the OD=0.1.

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. Add 6µl of AHL stock solution to 600µl of cell samples, to a final concentration of 1uM.

Day 2

Equipment

• Water bath @ 37°C
• Stripettes
• Well-plate x1
• Stop watch

Reagents

• LB medium
• E.coli culture with transformed plasmid
• Ampicillin stock (50 mg/ml)
• 10μM AHL stock
• 100μM AHL stock
• GFP stock solution
• ddH2O

Protocol

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)

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 Xµl of stock concentration of AHL to the respective wells as shown.
2. Incubate it at 37oC for 4 hours.
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)

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

Schematic

Well Test Construct Stock Volume (ul) AHL (ul) Final [AHL] A1 pTet-LuxR-pLux-GFP + 1μM AHL 200 2 10-6M A2 pTet-LuxR-pLux-GFP + 1μM AHL 200 2 10-6M A3 pTet-LuxR-pLux-GFP + 1μM AHL 200 2 10-6M A5 LB-Amp Media 200 0 0 A6 LB-Amp Media 200 0 0 A7 LB-Amp Media 200 0 0 B1 pTet-LuxR-pLux-GFP 200 0 0 B2 pTet-LuxR-pLux-GFP 200 0 0 B3 pTet-LuxR-pLux-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 C5 Diluted GFP Solution 200 0 0 C6 Diluted GFP Solution 200 0 0 C7 Diluted GFP Solution 200 2 0