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

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

1. Initial Promoter Testing - IN VIVO

1.1 Initial testing for Cell by Date

Overall Status
Results being uploaded...
Construct	Status
pTet-GFP	Tested - 16/08/2007
pT7-GFP	Planned - 17/08/2007
pCI-GFP	Waiting for construct

Constructs: pTet-GFP, pT7-GFP, pcI-GFP

Test to see if construct will express in vivo. Experiments carried out at 30°C in an incubator.

Aims:

• To determine if construct expresses in vivo
• To get approximations of: life span, response time and rate of GFP synthesized.
• To determine whether the constructs actually work before testing them in vitro.

	Protocol
Results

[+] Constant Conditions

• 50μl in vitro chassis
• DNA concentration
• Temperature stable at 30°C

[+] Variables

• Rate of GFP synthesis
• Life span of chassis
• Response time

[+] Sampling

• Every 5 minutes.

Repetition:

• 3 repeats

[+] Controls

• Negative Control: E.Coli cultures without GFP expressing machinery
• Positive Control: Diluted GFP in well

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1.2 Initial testing for Infector Detector

Status
Planned for 20/8/07	INCOMPLETE

Construct: pTet - LuxR - pLux - GFP

Test to see if construct will express in vivo. Experiments carried out in incubator at 30^oC at a range of inducer concentrations. To do this, we induce E. coli cells transfected with the construct with known concentrations of AHL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution.

	Protocol
Results

Aims:

• To determine if construct expresses GFP in vivo
• To determine the rate of change of protein production relative to concentration of AHL
• To determine whether the constructs need to be optimised before testing in vitro.

[+] Constant Conditions

• Temperature stable at 30°C

[+] Variables

• AHL concentrations
• Rate of GFP synthesis
• Life span of chassis
• Response time

[+] Sampling

• Every 5 minutes.

Repetition:

• 3 repeats

[+] Controls

• Negative Control: In vivo system with no AHL added
• Positive Control: In vivo system with a large amount of AHL added.

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2. Initial Promoter Testing - IN VITRO

2.1 Initial Testing for Cell by Date

Status
Planned for 22/8/07	INCOMPLETE

Constructs: pTet-GFP, pT7-GFP, pcI-GFP
.

	Protocol
Results

Aims:
Experiment 1 :

• Determine the DNA concentration and purity. From this determine volume of the DNA needed to add to the in vitro expression systems.

Experiment 2:

• To determine which constructs for cell by date expresses in vitro
• To test the pTet and pcI constructs in commercial S30 E.coli cell extract and home made S30 extract
• To test the pT7 construct in commercial S30 T7 cell extract and home made S30 extract.
• To investigate optimum volume of home made S30 extract
• To investigate the differences between home made and commercially bought S30 extract. This is in terms of rates of expression, length of expression and total output.

Status:

• DNA Constructs: Purified samples of pTet+pT7 available Friday, pcI available next week.
• T7 Commercial extract arrived
• E.coli commercial extract to arrive Friday
• Home made extract to be completed on Tuesday - delays from growth of E.coli cells and limitations of when we have access to the french press.

[+] Constant Conditions

• 2ug DNA added
• 30°C

[+] Variables

• Type of cell extract used : Commercial or home made
• For home made extract we will vary the volumes used

[+] Sampling

• Every 5 minutes initially however, after first few readings, this will be change based upon readings

Repetition:

• 2 repeats

[+] Controls

• Negative Control: In vitro system only to measure background fluorescence
• Positive Control: In vitro system with purified GFP added

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2.2 Initial testing for Infector Detector

Status
Planned for 21/8/07	INCOMPLETE

Construct: pTet - LuxR - pLux - GFP

Test to see if construct will express in vitro. Experiments carried out at incubator at 30^oC and under inducer concentration that was shown to give a high induction in vivo.

	Protocol
Results

Aims: Aims:
Experiment 1 :

• Determine the DNA concentration and purity. From this determine volume of the DNA needed to add to the in vitro expression systems.

Experiment 2:

• To determine if the infector detecter construct expresses in vitro
• To Test the construct in home made and in commercial e.coli cell extract

[+] Constant Conditions

• 30 ^oC
• DNA added 2μg

[+] Variables

• Type of cell extract used : Commercial or home made
• AHL concentration added: 1uM and 0.1uM

[+] Sampling

• Every 5 minutes initially and there after adjust sampling accordingly.

Repetition:

• 2 repeats

[+] Controls

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

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