IGEM:IMPERIAL/2007/Projects/Cell by date/Implementation: Difference between revisions

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===3) Operating Temperature Range===

Revision as of 04:13, 13 August 2007

Cell by Date: Implementation



Lab Notebook

  • Week 5 (6 Aug): Phase 1
  • Week 6 (13 Aug): Phase 1
  • Week 7 (20 Aug):
  • Week 8 (27 Aug):
  • Week 9 (3 Sep):
  • Week 10 (10 Sep):

<calendar> name=iGEM:IMPERIAL/2007/Notebook date=2007/09/15 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>


Phase 1: Initial Testing

1) Initial Testing

Constructs: pTet-GFP, pT7-GFP, pcI-GFP
Test to see if construct will express in vitro. Experiments carried out under room temperature 25°C.
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:

  • 25 oC
  • 50μl in vitro chassis
  • DNA concentration

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


Phase 2: Characterizing Specific Construct

1) Calibration Curve for GFP

Test to determine the relationship between fluorescence and in vitro concentration of GFP. To test this purified samples of known [GFP] are added to in vitro chassis and the fluorescence measured. From this a calibration curve of [GFP] vs Fluorescence can be made. This can be used for data analysis to convert fluorescence into a [GFP].
Aims:

  • To determine [GFP] vs Fluorescence

Constant Conditions:

  • 50μl in vitro chassis
  • DNA added
    • Do we need DNA, as it may absorb some of the fluorescence
  • 25oC

Variables:

  • Independent variables :
    • [GFP] added
  • Dependent variables:
    • Fluorescence

Sampling:

  • Measure after addition of GFP to minimize degradation of GFP

Repetition:

  • 3 repeats

Controls:

  • Negative Control:
    • No GFP is added to an in vitro chassis


2) Degradation Time of GFP

Test the half life of GFP protein in an in vitro chassis. To test this a purified sample of known [GFP] are added to an in vitro chassis, then fluorescence will be measured at regular time intervals. The fluorescence will be converted into GFP molecules using the calibration curve. This will give; degradation of GFP as a function of time, from this the half life of GFP can be obtained. In addition, temperature may affect the half life of GFP and so the half life will be measured for an appropriate temperature range.
Aims:

  • To determine the half life of GFP for a range of temperatures

Constant Conditions:

  • 50μl in vitro chassis
  • GFP added....

Variables:

  • Independent variables :
    • Temperature range:4oC, 15oC, 25oC, 30oC, 37oC, 50o
    • Range may change based upon the initial testing; will only test ranges that in vitro is stable at.
  • Dependent variables:
    • Degradation of GFP

Sampling:

  • Every 5 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: No GFP is added to an in vitro chassis
  • Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature

(CHECK)


3) Operating Temperature Range

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