Initial Testing for DNA constructs In Vivo

Aims

• To test to see if the DNA constructs from the registry are viable. This is done in vivo.
• The constructs are pTet-GFP, pT7-GFP and pcI-GFP.

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 - This concentration was unknown, however because we only wanted it as a positive control to show there was fluorescence and that the flurometer could measure it

Protocol

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

• Well-plate x1
• Fluorometer
• Gilson pipettes 1000 and 200
• Eppendorf tubes

Reagents

• ddH2O
• GFP standard solution

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)
2. Place the tube on ice till it is ready to be used.

Loading Plate

1. Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. (Follow the schematic as shown)
2. Three wells to be filled with 200µl of media to measure the absorbance background.
3. Standard GFP solution added as a positive control.
4. 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)

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