IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 2.1.1

Experiments - 14/09/2007

Experiments to be carried out are to determine the working temperature range of the pTet-GFP construct, in-vitro. Each temperature will be tested over a period of 6 hours, as it is expected that the system will respond within about 3-4 hours to a change in the ambient temperature. The evaporation of the samples will be taken into account when analysing the data.

Aims

• To determine if the construct expresses in vitro at temperatures: 8°C, 15°C, 25°C, 30°C
• To determine the rate of GFP produced at each temperature range by measuring fluorescence reading over a period of 6 hours
• To determine how long the system can last at 4°C (fridge temperature), after it has expressed in a particular temperature for 6 hours
• To see if evaporation is significant to our results over a period of 6 hours

Equipment

• Fluorometer + PC
• Water bath in cold room at 8°C/15°C
• 25°C/30°C water bath
• 4 Fluorometer plates
• 4 Sealing plate mats
• Gilson pipettes 200, 20, 10
• Eppendorf Tube x 4
• Plate Centrifuge
• 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

Protocols

1. First collect all equipment and reagents and ensure that the fluorometer and the PC connected has a data collection protocol installed.
2. Place each of the 96 well plates together with their plate mats in their respective incubators so as to get them to the appropriate temperature before the experiments start.
3. For the cell extract, get the following out of the cell extract kit:
   • A.A's from kits
   • 2xPremix tubes (60ul each)
   • 2xS30 tubes (45ul each)
     
4. For Each Temperature Carry out the following Procedure:
   
5. Commercial E.coli Cell Extract:
   1. First prepare a complete amino acid mixture for both extract solutions: Add the 10µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 20µl. 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.
   2. Take an eppendorf tube and add 20µl of the E.coli complete amino acid mixture.
   3. Add 80µl of S30 Premix Without Amino Acid.
   4. Then add 60µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench.
   5. Vortex the tubes to mix thoroughly and place the tubes of E.coli commercial extract in the appropriate incubator for ten minutes. Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
6. Separate 60µl of maxipreped DNA plasmid into an eppendorf tube and place them in their respective incubators as well.
7. Separate 20ul of maxipreped DNA of empty vector into separate tubes.
8. Fill some of the centre and side wells, which have no cell extract and DNA mixture in them, with 60ul of water. These samples will be used to measure the amount of evaporation of the samples.

Loading Plate

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. Tap down the top of the lid to bring down any solution to bottom of the well. Remove lid off the 96 well plate and place in the fluorometer. Create a file name insert temp under: D:\IGEM\INSERT DATE\CBD\ OTR. Export the data here. Each file should be named as the following: construct-temp-time-date This measurement will give a back ground fluorescence measurement and can be used as our time zero data. 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. Place lid back on and place back in the respective incubators. After 30 minutes of incubation measure the fluorescence by repeating procedure 3-4 above. Keep taking measurments like this every 30 mins, until 6 hours have elapsed since the first reading, with DNA. Before each measurement be careful to remember to tap down the solution and to remove the lid before placing in the fluorometer.

After the last reading, check the samples under a UV light to see if the fluorescence reached is visible or not. Turn the lights off in the room before proceeding with this. Now measure the amount of water left in the wells to see if evaporation affected the results or not.

Leave the plates incubated in 4oC over a period of two nights. This will allow to test for the lifespan of the DNA ans cell extract mixture, after it has reacted (see application specs).

Day 2:

Experiment - 30/08/2007

• Temperatures will be carried out over a period of a week. Two temperatures will be carried out at once. See lab note book for exact time tabeling.
• For each temperature we will be carrying out two experiments, one will start at 10am one will start at 8pm. This staggering is to narrow the gap over night that we cannot measure.
• The protocol below describes what should be done for one experiment, for the last experiment repeat the protocol, but do not measure in the fluorometer.

Aims

• To determine if construct expresses in vitro at temperatures of: 10°C, 15°C, 20°C, 25°C, 30°C, 37oC, 45°C
• To determine how long the system can last (when GFP is no longer produced) at each temperature.
• To determine the rate of GFP produced at each temperature range by measuring fluorescence reading over a period of 30 hours.

Equipments

• Fluorometer + PC
• Water bath in cold room at 10°C/15°C/20°C
• 30°C/45°C water bath
• 37°C incubator
• 6 Fluorometer plates (black)
• 6 Sealing plate mats
• Gilson pipettes 200, 20, 10
• Eppendorf Tube x 4
• Plate Centrifuge
• 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
• Commercial S30 T7 extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl T7 S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• Nuclease Free water
• GFP solution

Steps

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 each of 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
   • 2xPremix tubes (60ul each)
   • 2xS30 tubes (45ul each)
4. For Each Temperature Carry out the following Procedure
5. Commercial E.coli Cell Extract: First prepare a complete amino acid mixture for both extract solutions: Add the 10μl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 20μl. 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:Take an eppendorf tube and add 20µl of the E.coli complete amino acid mixture. Then add 80µl of S30 Premix Without Amino Acid. Then add 60µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench.
7. Vortex the tubes to mix thoroughly and place the tubes of E.coli commercial extract in each incubator for ten minutes.
8. Separate 80μl of midipreped DNA plasmid into an eppendorf tube and place them in their respective incubators as well.
9. Separate 20ul of midipreped DNA of empty vector into separate tubes.
10. Any left over premix or cell extract should be returned to the freezer in biochemistry level 5 and labeled with new volumes.

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\CBD\ OTR. 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.

Schematic

Plan for Temperature Testing Day 1-3

Well Test Construct In vitro chassis Vol of in vitro chassis (ul) A1 pTet Commercial E.coli extract 100µl-DNA volume A2 pTet Commercial E.coli extract 100µl-DNA volume A3 pTet Commercial E.coli extract 100µl-DNA volume B1 pTet Commercial T7 extract 100µl-DNA volume B2 pT7 Commercial T7 extract 100µl-DNA volume B3 pT7 Commercial T7 extract 100µl-DNA volume C1 pT7 Commercial E.coli extract 100µl-DNA volume C2 pT7 Commercial E.coli extract 100µl-DNA volume C3 pT7 Commercial E.coli extract 100µl-DNA volume G1 pT7 Diluted GFP 110µl G2 pT7 Commercial E.coli extract 100µl-DNA volume G3 pT7 Commercial T7 extract 100µl-DNA volume