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

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1. Initial Promoter Testing - IN VIVO


1.1 Initial testing for Cell by Date

Overall Status
Results uploaded !
Construct Status
pTet-Gfp Tested - 16/08/2007
pT7-Gfp Tested - 17/08/2007
pcI-Gfp ABORTED - Problem with Biobricks


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


Test to see if construct will express in vivo. Experiments carried out at 37°C in an incubator.


Aims:

  • To determine if construct expresses in vivo


Protocol
Results


[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls

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1.2 Initial testing for Infector Detector

Overall Status
Results uploaded !
Construct Status
pTet-LuxR-pLux-GFP COMPLETED


Construct: pTet-LuxR-pLux-GFP

Test to see if construct will express in vivo. Experiments carried out in incubator at 37oC at a constant inducer concentration. To do this, we induce E. coli cells transfected with the construct with a 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


[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls

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2. Initial Promoter Testing - IN VITRO


2.1 Initial Testing for Cell by Date

Overall Status
Results uploaded !
Construct Status
pTet-Gfp Initial Testing - 21/08/2007
pT7-Gfp Initial Testing - 21/08/2007
pcI-Gfp ABORTED - Problem with Biobricks




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


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.
Protocol
Results

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.


[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls

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2.2 Initial testing for Infector Detector

Overall Status
Results uploaded !
Construct Status
pTet-LuxR-pLux-GFP COMPLETED




Construct: pTet-LuxR-pLux-GFP



Protocol
Results


Aims:
Test to see if construct will express in vitro. Experiments carried out at incubator at 37oC and under an inducer concentration that was shown to give a high induction in vivo.


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 infecter detector construct expresses in vitro
  • To Test the construct in home made and in commercial e.coli cell extract



[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls

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3. Calibration Experiments


3.1 Investigate optimum counting time for fluorodetector


Status
Results uploaded !
Planned for 21/08/2007 COMPLETED


Construct: pTet-GFP


A test to see what is the optimum counting time to have during our experiments. The counting time is the time the fluorometer detector stays on top of each well. A small window time will only account for very discrete levels of fluorescence. These might include sudden spikes of radiation since fluorescence is not a uniform process. Hence we will get variation between samples of equal expression rates. A larger counting time results in a larger window size and hence a more average reading is taken from each sample smoothing out the variation due to the randomness of fluorescence emition.
Care must be taken however because larger counting times will lead to faster fluorescence bleaching. A comprimise between the two must be found.

Protocol
Results


Aims:
Determine the optimum counting time for the fluorometer. Avoid fluorescence bleaching.



[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls

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3.2 Investigate the efficiency of a 1:1 mixture of commercial and homemade extract


Status
Planned for 29/08/2007 COMPLETED


Construct: pTet-GFP


This experiment is done to test if the commmercial cell extract and pre-incubation mix, if mixed in a ratio of 1:1 with the home made cell extract and reaction buffer, will produce sufficiently satisfying results to be used for carrying out the rest of the experiments for CELL BY DATE and INFECTOR DETECTOR.

Protocol
Results


Aims:
To determine the working efficiency of the mixture of commercial and home-made cell extracts.



[+] Constant Conditions

[+] Variables

[+] Sampling

[+] Controls

Show All Details
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