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Latest revision as of 03:17, 27 October 2007

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Wet Lab: Protocols: Testing of Purified LuxR

Aims:
To confirm that the LuxR that we have successfully purified is functional. We need the LuxR to still have DNA specificity and be functional.

Status:

• To be carried out 27/09/2007

Equipment

• Fluorometer + Connected PC
• 25°C incubator
• 1 Fluorometer plate (black)
• Sticky seal tape
• Gilson pipettes p200 p20 p10
• Eppendorf Tubes
• Stopwatch

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
• DNA pLux-GFP from midiprep

Protocol

1. First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
2. Place the 96-well plate 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:
   
   1. First prepare a complete amino acid mixture for the extract solution: Add the 7.5µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 15µl. Each amino acid minus mixture is missing one type of amino acid.
5. Add 60µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
6. Then add 45µl of S30 Cell Extract Circular too. This mixture is for all the samples. Label the tube. Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
7. Now combine the Cell Extract and AA to give a final volume : 120µl and incubate in the water bath set at 25°C.
8. Remove 9µl from the 1000nM stock solution of AHL and pipette into an eppendorf tube. Incubate the eppendorf tube in the 25°C water bath.
9. Prepare the different DNA concentrations for construct 1. We need to prepare two solutions for construct 1, one solution with 2ug in 17ul and one with 2ug in 16ul. The two solutions are used because for one solution we are adding LuxR (1ul) and so we compensate by decreasing the water added to the DNA dilution.
   • Construct 1 (16ul Maxi DNA)x2
   • Construct 1 16ul Maxi DNA + 1ul Nuclease Free Water

Loading Plate

1. Now we can combine the reaction and load into the wells. We need to ensure that this is done as quickly as possible and that it is timed from the addition of the DNA to solution:
   1. 43ul of cell extract + AHL and 17ul DNA solution 2 into a tube
   2. 86ul of cell extract + AHL and 32ul DNA solution 1 into a tube and 2ul LuxR protein
2. Then load up the following into the three wells, 60ul each.
3. Place the plate in the fluorometer to measure its initial fluorescence reading.
4. After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
5. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
6. Repeat the reading every 30 minutes, for 6 hours.