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Protocols for DNA concentration experiments

Experiments to be carried out are to determine the optimum concentration of the ID construct, in-vitro, so that we get the highest level of protein expression after a period of 6hours. The constructs to be tested is pTet-luxR-pLux-GFP.

The concentrations of DNA that will be tested are: 1, 2, 4 and 6µg. For ID construct, Each concentration of DNA will be tested over a period of 6 hours at 25°C, as it is expected that the system will respond within about 2-3 hours to AHL (50nM).

Aims

• To determine the concentration of pLux construct for which the response to AHL (50nM) induction is optimum, in terms of the reponse time and the output fluorescence at the end of the experiment time.

Equipment

• Fluorometer + PC
• 25°C water bath
• Fluorometer plate (black)
• Sticky seal tape
• Gilson pipettes 200, 20, 10
• Eppendorf Tubes x 7
• Stopwatch
• Foil

Reagents

• Commercial S30 E.coli extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• Nuclease Free water x1ml
• DNA pTet-LuxR-pLux-GFP from midiprep

Preparation of reactions

1. First collect all equipment and reagents and ensure that the fluorometer and the PC connected has a data collection protocol installed.
2. Place one of the 96-well plates into the 25°C water bath.
3. For the cell extract, get the following out of the cell extract kit:
   • A.A's from kits
   • Premix tube
   • S30 tubes
4. To prepare the commercial E.coli Cell Extract, carry out the following procedure, two times:
   
   1. First prepare a complete amino acid mixture for the extract solution: Add the 25µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 50µl. Each amino acid minus mixture is missing one type of amino acid.
   2. Take an eppendorf tube and add the 50µl of the E.coli complete amino acid mixture.
   3. Add 200µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
   4. Then add 150µl of S30 Extract Circular too.
   5. The final volume of cell extract is: 400µl
   6. Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
5. Each cell extract will be used to test one of the constructs. Label the tubes, identifying which construct it will be used for.
6. Incubate cell extract mixture for ID in the water bath set at 25°C.
7. Get 30µl out of the 1000nM stock solution of AHL and put in to the eppendorf tube with the cell extract for the pLux construct. This will give a AHL concentration of 50nM in the final 60µl of the samples. Incubate the eppendorf tube in the 25°C water bath.
8. Prepare the different DNA concentrations for pLux construct (concentration of pLux DNA = 460ng/µl):
   1. Concentration 1 = 1µg: Add 4.4µl of DNA in 29.6µl nuclease free water.
   2. Concentration 2 = 2µg: Add 8.8µl of DNA in 25.2µl nuclease free water.
   3. Concentration 3 = 4µg: Add 17.4µl of DNA in 16.6µl nuclease free water.
   4. Concentration 4 = 6µg: Add 26µl of DNA in 8µl nuclease free water.
9. This will give a total volume of 34µl of each DNA concentration. Put each DNA into a seperate, labeled eppendorf tube and place them in the 25°C water bath.

Loading Plate

1. Take the plate out of the incubation.
2. For the pLux construct:
   1. Follow the schematic for the plate 1 (25°C water bath) and begin by loading 43µl of the in vitro expression system with AHL into the right wells.
   2. Tap down the top of the plate to bring down any solution to bottom of the well.
   3. Then add 17µl of purified DNA sample to each well, as indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
   4. Add 17µl of nuclease free water into the two negative control wells, as shown in the schematics.
3. After the DNA and the cell extract mixtures have been put into their respective wells, load the program on the PC to measure the fluorescence in the right wells.
4. Create a file with name referring to the temperature of the plate, under: D:\IGEM\INSERT DATE\ID\ OTR. The data from the fluorometer will be exported here.
5. Each file with the reading should be named as the following:
   • construct-temp-time-date
6. Place the plate in the fluorometer to measure its initial fluorescent reading.
7. After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
8. Before placing it in the water bath, wrap aluminium foil around them to prevent photobleaching.
9. Measure the temperature every 30 minutes for each temperature, for 6 hours.

Schematic

Plate 1

Well	Test Construct	Concentration of DNA	In vitro chassis
E5	Nuclease Free Water + AHL (Negative control)	0µg	Commercial E.coli extract
E7	Nuclease Free Water + AHL (Negative control)	0µg	Commercial E.coli extract
C3	pTet-luxR-pLux-GFP + AHL	1µg	Commercial E.coli extract
C5	pTet-luxR-pLux-GFP + AHL	1µg	Commercial E.coli extract
C7	pTet-luxR-pLux-GFP + AHL (positive control)	2µg	Commercial E.coli extract
C9	pTet-luxR-pLux-GFP + AHL (positive control)	2µg	Commercial E.coli extract
D4	pTet-luxR-pLux-GFP + AHL	4µg	Commercial E.coli extract
D6	pTet-luxR-pLux-GFP + AHL	4µg	Commercial E.coli extract
D8	pTet-luxR-pLux-GFP + AHL	6µg	Commercial E.coli extract
D10	pTet-luxR-pLux-GFP + AHL	6µg	Commercial E.coli extract