IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase1: Difference between revisions
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=1. Initial Promoter Testing - In Vivo= | |||
==1.1 Initial testing for Cell by Date== | |||
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==1.2 Initial testing for Infector Detector== | |||
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*Positive Control: In vitro system with purified GFP added | *Positive Control: In vitro system with purified GFP added | ||
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Revision as of 06:42, 14 August 2007
1. Initial Promoter Testing - In Vivo
1.1 Initial testing for Cell by Date
| Status | |
| Planned for 15/8/07 | INCOMPLETE |
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.
| Protocol | |
| Results | |
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.
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
1.2 Initial testing for Infector Detector
| Status | |
| Planned for 15/8/07 | INCOMPLETE |
Construct: pTet - LuxR - pLux - GFP
Test to see if construct will express in vivo. Experiments carried out in incubator at 30oC 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.
2. Initial Promoter Testing - In Vitro
2.1 Initial Testing for Cell by Date
| Status | |
| Planned for 15/8/07 | INCOMPLETE |
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.
| Protocol | |
| Results | |
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.
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: In vitro system only
- Positive Control: In vitro system with purified GFP added
2.2 Initial testing for Infector Detector
| Status | |
| Planned for 15/8/07 | INCOMPLETE |
Construct: pTet - LuxR - pLux - GFP
Test to see if construct will express in vitro. Experiments carried out at incubator at 30oC and under inducer concentration that was shown to give a high induction in vivo.
| Protocol | |
| Results | |
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:
- 25 oC
- 50μl in vitro chassis
- DNA added 2μg
- AHL concentration 1000nM
Variables:
- Independent variables :
- Dependent variables:
- Rate of GFP synthesis
- Life span of chassis
- Response time
Sampling:
- Every 5 minutes.
Repetition:
- 3 repeats
Controls:
- Negative Control: In vitro system with no AHL added
- Positive Control: In vitro system with purified GFP added
