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
• 2000, 1000, 400, 300, 200 and 100nM AHL stock solutions
• pTet-LuxR-pLux-GFP mut 3b 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 (150ul)
4. Prepare the following cell extract mixture for all 21 reactions(one extra reaction):
   • First add 420µl of the premix solution into a labelled eppendorf.
   • Then add 315µl of the S30 cell extract mixture to S30 Premix Without Amino Acid
   • Then prepare a complete amino acid mixture: Add 52.5μl of two amino acid minus mixtures into the eppendorf with the premix and S30 solution. 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.
   • Vortex the tubes to mix thoroughly
   • Any left over premix or cell extract should be returned to the freezer in biochemistry level 5 and labeled with new volumes.
5. Prepare the following dilution to get DNA concentration of 4µg:
   • Put 208.8µl of pLux DNA into a labelled eppendorf
   • Add 199.2µl of nuclease free water into the DNA
   • This will give a total volume of 408&micr;l, which is enough for all 18 samples with DNA in them

Loading Plate

1. First read the background fluorescence of the 96-well plate using the fluorometer.
2. Choose suitable wells, with minimum fluorescence (30-40 au) to put the samples in. Don't use the wells at the edges and avoid putting samples in consecutive wells.
3. 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.
4. Tap down the top of the lid to bring down any solution to bottom of the well.
5. 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
6. This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
7. Then to begin the reaction add 17μl of purified DNA sample to each well indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
8. Place on and place back in the respective incubators.
9. 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.
10. 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