Imperial/Wet Lab/Protocols/ID2.1

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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 (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-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 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. Place the plate in the fluorometer to measure its initial fluorescent reading.
  9. After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
  10. Start on the next plate, and repeat procedures 2-6.
  11. Place the plate in the 37oC water bath.
  12. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
  13. Stagger the start of both the plates by around 5 minutes.
  14. 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


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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