From 2007.igem.org

Protocols for DNA concentration experiments

Experiments to be carried out are to determine the optimum concentration of the ID and CBD constructs, in-vitro, so that we get the highest level of protein expression after a period of 6hours. The two constructs to be tested are pTet-luxR-pLux-GFP and pTet-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). For ID the samples will be kept at 37°C. The evaporation of the samples will be taken into account when analysing the data.

Aims

• To determine the concentration of pLux construct for which the response to AHL (at 50nM) being induced is optimum, in terms of the reponse time and the output fluorescence at the end of the experiment time.
• To determine the concentration of pTet construct for which output 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
• Gilson pipettes 200, 20, 10
• Eppendorf Tubes x 7
• Stopwatch
• Foil
• Clear tape

Reagents

• S30 E.coli Cell Extract
• Nuclease Free water
• DNA

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 96well plates into the 25°C water bath and the other in the 37°C incubator.
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 and the one for CBD in the 37°C incubator.
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.
10. Prepare the different DNA concentrations for pTet construct(concentration of pTet DNA = 500ng/µl):
    1. Concentration 1 = 1µg: Add 4µl of DNA in 36µl nuclease free water.
    2. Concentration 2 = 2µg: Add 8µl of DNA in 32µl nuclease free water.
    3. Concentration 3 = 4µg: Add 12µl of DNA in 28µl nuclease free water.
    4. Concentration 4 = 6µg: Add 16µl of DNA in 16µl nuclease free water.
11. This will give a total volume of 40µl of each DNA concentration. Put each DNA into a seperate, labeled eppendorf tube and place them in the 37°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. For the pTet construct:
   1. Follow the schematic for the plate 2 (37°C incubator) and begin by loading 40µl of the in vitro expression system 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 20µl 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 20µl of nuclease free water into the two negative control wells, as shown in the schematics.
4. Put 60µl of water into some empty wells in the middle of each plate. These will be used to check for evaporation.
5. 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.
6. Create a file with name referring to the temperature of the plate, under: D:\IGEM\INSERT DATE\ID\ OTR , for ID and under D:\IGEM\INSERT DATE\CBD\ OTR , for CBD construct. The data from the fluoreometer will be exported here.
7. Each file with the reading should be named as the following:
   • construct-temp-time-date
8. While the program loads, get the plate out of the water bath/incubator and wipe off the water on it.
9. Take a reading in the fluorometer. Before each measurement remember to tap down the solution and to remove the clear tape on it before placing in the fluorometer.
10. As soon as the reading has been taken, unload the plate and place the clear tape on the plate and place back in the water bath. Cover the plate with foil to prevent the DNA from getting bleached due to light. Make sure that the plate is not outside the water bath for longer than 5mins. Remember to close the plate holder of the fluorometer after each reading.
11. After 1 hour of incubation, load the program on the PC again, to measure the fluorescence in the right wells.
12. Take another fluorescence reading, repeating steps 6-13.
13. Take a reading similarly every hour, until 6 hours have elapsed since the first reading.
14. After the last reading, measure the amount of water left in the wells (with no cell extract mixture) to check the amount of fluid that has evaporated, using a gilson pipette.
15. Wash off the plates with 70% ethanol and rinse with distilled water

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

Plate 2

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

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