User:Maira Tariq/sandbox

Protocols for experiments to determine sensitivity to AHL of the ID

Aims

• To determine the response of the pTet-LuxR-pLux-GFP construct, in-vitro, for different concentrations of AHL
• To determine the response time of the construct in-vivo, for different AHL concentrations

Day 1

Equipment

• Eppendorf Tubes
• Gilson pipettes p1000, p200, p20, p10

Reagents

• AHL stock solution of 1mM
• Nuclease free water

Protocols

• Using stock solution of 1mM of AHL, carry out the following dilutions using nuclease free water:
  1. Add 2ul of 1mM AHL to 998ul of water in an eppendorf tube, making up a 1000µl stock of 2000nM AHL
  2. Remove 500µl from the 2000nM solution and put into another eppendorf tube with 500µl of water. This will give a 1000µl solution of 1000nM AHL
  3. Remove 400µl from the 1000nM AHL solution and put in 600µl of water. This makes a 1000µl solution of 400nM AHL.
  4. Remove 151.5µl from the 1000nM AHL and put into an eppendorf with 348.5µl of water. This gives 500µl of 300nM AHL
  5. Remove 500µl of 400nM AHL and put into an eppendorf with 500µl water. This makes 1000µl of 200nM AHL
  6. Remove 500µl from 200nM AHL and put into an eppendorf tube with 500µl of water. The solution made is 1000µl of 100nM AHL.

Day 2

Equipments

• Fluorometer + PC
• Water bath in cold room at 25°C
• 1 Fluorometer plates (black)
• Clear tape
• Gilson pipettes 200, 20, 10
• Eppendorf Tube x 4

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
• AHL solutions A B C
• DNA construct

Protocols

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 plates together with their plate mates in their respective incubators so as to heat them up to the appropriate temperature before the experiments start.
3. For the next step of the go to the biochemistry level 5 and remove:
   • A.A's from kits
   • Premix tubes (140ul)
   • S30 tubes (105ul)
4. For each AHL concentration Tested Prepare the following
5. Commercial E.coli Cell Extract: First prepare a complete amino acid mixture for both extract solutions: Add the 17.5μl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 35μl. Each amino acid minus mixture is missing one type of amino acid, and so by combining two solutions we are complementing each solution for the missing amino acid. Place eppendorf in a rack on bench.
6. Commercial E.coli Cell Extract:Add all of the E.coli complete amino acid mixture to S30 Premix Without Amino Acid and 45µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench.
7. Vortex the tubes to mix thoroughly and place 40ul into each well-B4, B6, C5, C7, D2, D4, and E8.
8. Place 20ul of midipreped DNA plasmid into each of the filled wells.
9. Any left over premix or cell extract should be returned to the freezer in biochemistry level 5 and labeled with new volumes.

Schematic

• Throughout our series of experiments we are reusing the 96 well plates. It is important to measure each plate before the schematic is determined, this is to prevent any contamination.
• Using the protocol above using wells near the edge should be avoided and the wells spread out throughout the plate
• Add 3ul of solution A to two wells B4 and B6. [AHL]=100nM
• Add 3ul of solution B to two wells C5 and C7. [AHL]=50nM
• Add 3ul of solution C to two wells D2 and D4. [AHL]=10nM
• Add 3ul of nuclease free water to well E8. [AHL]=0nM

Loading Plate

1. Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
2. Tap down the top of the lid to bring down any solution to bottom of the well.
3. Remove lid off the 96 well plate and place in the fluorometer. Create a file name insert temp under: D:\IGEM\INSERT DATE\ID\ 25oC. Export the data here. Each file should be named as the following:
   • construct-temp-time-date
4. This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
5. Then to begin the reaction add 20μl of purified DNA sample to each well indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
6. Place lid back on and place back in the respective incubators.
7. After 10 minutes of incubation measure the fluorescence by repeating procedure 3-4 above. This initial measurement of 10 minutes should be carried on for 1 hours or until it appears GFP production levels off.
8. Before each measurement be careful to remember to tap down the solution and to remove the lid before placing in the fluorometer.

Well Test Construct Stock Volume (ul) AHL (ul) Final [AHL] B4 pTet-LuxR-pLux-GFP 60 3ul 100nM B6 pTet-LuxR-pLux-GFP 60 3ul 100nM C5 pTet-LuxR-pLux-GFP 60 3ul 50nM C7 pTet-LuxR-pLux-GFP 60 3ul 50nM D4 pTet-LuxR-pLux-GFP 60 3ul 10nM D6 pTet-LuxR-pLux-GFP 60 3ul 10nM E8 pTet-LuxR-pLux-GFP 60 0 0