IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase1

• Introduction
• Phase 1
• Phase 2
• Phase 3
• Notes

Initial Promoter Testing

1) Initial Testing for Cell by Date

Constructs: pTet-GFP, pT7-GFP, pcI-GFP
Test to see if construct will express in vitro. Experiments carried out at 30°C in an incubator.
Aims:

• To determine if construct expresses in vitro
• To get approximations of: life span, response time and rate of GFP synthesized.
• To determine whether the constructs or the in vitro need to be optimised.

Conditions:
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• 50μl in vitro chassis
• DNA concentration

Variables:
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• Temperature change 30°C
• Rate of GFP synthesis
• Life span of chassis
• Response time

Sampling:
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• Every 15 minutes.

Repetition:

• 3 repeats

Controls:
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• Negative Control: In vitro system only
• Positive Control: In vitro system with purified GFP added

2) Initial testing for Infector Detector

Construct: pTet - LuxR - pLux - GFP
Test to see if construct will express in vitro. Experiments carried out under room temperature 25^oC and under inducer concentration that was shown to give a high induction in vivo.
Aims:

• To determine if construct expresses in vitro
• To get approximations of: life span, response time and rate of GFP
• To determine whether the constructs or the in vitro need to be optimised.

Constant Conditions:
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• 25 ^oC
• 50μl in vitro chassis
• DNA added 2μg
• AHL concentration 1000nM

Variables:
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• Independent variables :
• Dependent variables:
  • Rate of GFP synthesis
  • Life span of chassis
  • Response time

Sampling:
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• Every 5 minutes.

Repetition:

• 3 repeats

Controls:
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• Negative Control: In vitro system with no AHL added
• Positive Control: In vitro system with purified GFP added

Equipment

• Incubator 30^oC
• Fluorometer
• Plate x1
• Plate Centrifuge
• 6x1.5ml tubes
• Gilson Pipettes
• Stop watch

Preparations of Chemicals and Reagents
After preparing all solutions preheat to 30^oC

1. Remove .... of our cell extract solution from storage solution
2. Now we need to prepare 50μl of the commercial E.coli and T7 cell extract solution. First we need to prepare complete amino acid mixture for both extract solutions: Add the 7.5μl volume of two amino acid minus mixtures for both kits
3. Take 3x1.5ml tube and add 5µl of the E.coli complete amino acid mixture
4. Take 3x1.5ml tube and add 5µl of the T7 complete amino acid mixture
5. To each 1.5ml tube add 20µl of S30 Premix Without Amino Acid
6. Add 15µl of S30 Extract Circular
7. Add ...nuclease-Free Water

Loading Plate

1. Follow the schematic for the plate adding everything but the DNA sample.
2. Place the top on the plate and place in the incubator. Leave for a few minutes to heat to 30^oC
3. Remove from incubator and centrifuge for 1 minute
4. Remove lid and Measure in the flourometer.
5. Then to begin the reaction add .... purified DNA sample.
6. Place lid back on and place back in the incubator at 30^oC

Collecting Data

1. After 5 minutes of incubation measure the fluorescence by repeating procedure 3-4 above.
2. Repeat measurements after every 5 minutes until the Fluorescence is constant
3. Before every measurement in the fluorometer spin the plates in a plate centrifuge

Schematic

Well Test Construct In vitro chassis A1 pTet Commercial E.coli extract A2 pTet Commercial E.coli extract A3 pTet Commercial E.coli extract A4 pTet Our S30 Cell extract A5 pTet Our S30 Cell extract A6 pTet Our S30 Cell extract A7 None Commercial E.coli extract A8 None Our S30 Cell extract B1 pT7 Commercial T7 extract B2 pT7 Commercial T7 extract B3 pT7 Commercial T7 extract B4 pT7 Our S30 Cell extract B5 pT7 Our S30 Cell extract B6 pT7 Our S30 Cell extract B7 None Commercial T7 extract B8 None Our S30 Cell extract