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

Experiment

Aims

• To determine if construct expresses in vitro at temperatures of: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
• To determine how long the system can last (when GFP is no longer produced) at each temperature.
• To determine the maximum rate of GFP produced at each temperature range by taking fluoresence reading over a period of time ( time frame to be determined by Friday 17th August)

Status

???

Equipments

• Fluorometer + PC
• Fridge at 4oC
• Water bath in cold room at 10oC/15oC/20oC
• 30oC water bath
• 37oC shaking incubator
• 50oC/60oC heating block
• 6 Fluorometer plates (black)
• 6 Sealing plate mats
• Gilson pipettes 200, 20, 10
• Eppendorf Tubes
• Plate Centrifuge
• Stopwatch

Reagents

• Our Prepared S30 extract: Optimised amount for experimentation to be decided after experiment 2.1
• 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
• MiiA water x1ml
• GFP solution (For this initial experiment does not need to be purified GFP, we just want to know we have the right filter and that our settings are adjusted to measuring GFP)

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 mats in their respective incubators so as to heat them up to the appropriate temperature before the experiments start.
3. Commercial E.coli Cell Extract: First prepare a complete amino acid mixture for both extract solutions: Add the 30μl volume of two amino acid minus mixtures into an labelled eppendorf to give a volume of 60μl. 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.
4. Commercial E.coli Cell Extract:Take a eppendorf tube and add 5µl of the E.coli complete amino acid mixture. Then add 20µl of S30 Premix Without Amino Acid. Then add 15µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench. Repeat the step for a total of 12 tubes.
5. Vortex the tubes to mix thoroughly and place the 4 tubes of E.coli commercial extract in each incubator for ten minutes.

• Separate 60μl of midipreped DNA plasmid into 3 separate tubes and place them in their respective incubators as well.

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. Remove from incubators and spin-down in centrifuge in plate centrifuge at 2000rpm for a few seconds. Spin down is the process of bringing down any solution on lid or side of well into the base of the well. Alternatively can tap 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 protocol 2-1 under: D:\IGEM\INSERT DATE\CBD\ OTR. Export the data here. If repeated measurements change the second number to suit repeat number, e.g. 2nd repeat protocol 2-2, 5th repeat protocol 2-5. Once the data collection is set up then initiate the measurements.
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 required volume of purified DNA sample to give 2µg to the wells 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 incubator at 30^oC
   
   
7. After 30 minutes of incubation measure the fluorescence by repeating procedure 3-4 above. This initial measurement of 30 minutes is to find out how fast GFP is being produced. After this initial measurement, the intervals should be reassessed and adjusted accordingly
8. Before each measurement be careful to remember to either spin down or tap down the solution and to remove the lid before placing in the fluorometer
9. Repeat the above steps until 6 hours after the DNA was first added.

Schematic

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