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

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==Phase 1: Initial Testing==
==Phase 1: Initial Testing==
==''Phase 1''==
===1)Initial Testing===
===1)Initial testing===
'''Constructs:''' pTet-GFP, pT7-GFP, pcI-GFP<br>
'''Construct:''' pTet - LuxR - pLux - GFP <br>
Test to see if construct will express ''in vitro''. Experiments carried out under room temperature 25&deg;C.<br>
Test to see if construct will express in vitro. Experiments carried out under room temperature 25<sup>o</sup>C and under inducer concentration that was shown to give a high induction in vivo.<br>
'''Aims:'''
'''Aims:'''
*To determine if construct expresses in vitro
*To determine if construct expresses ''in vitro''
*To get approximations of: life span, response time and rate of GFP
*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.
*To determine whether the constructs or the ''in vitro'' need to be optimised.
'''Conditions:'''
'''Conditions:'''
<br>
<br>
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*25 <sup>o</sup>C
*25 <sup>o</sup>C
*50μl in vitro chassis
*50μl in vitro chassis
*DNA concentration .......
*DNA concentration
*AHL concentration 1000nM
 
}}
}}
'''Variables:'''
'''Variables:'''
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}}
}}
<br>
<br>
===Materials & Methods===
*'''[[IGEM:IMPERIAL/2007/DNA_Constructs|DNA constructs]]''' to be ligated into plasmid.
*'''[[IGEM:IMPERIAL/2007/Projects/General Protocols | General Protocols]]''' for digestion, ligation, gel electrophoresis, gene cloning, electroporation, competent cells etc. and the materials required for each.
===Results===
'''8th Aug'''<br>
* Selecting transformed cells
** Competent cells were induced to uptake the plasmids.
** Culture is then plated on antibiotic plates to select for uptake.
'''9th Aug'''<br>
* Amplification of BioBricks
** Colonies that form on antibiotic plates are isolated and incubated o/n to amplify the BioBricks.
** pBad promoter and pBad construct did not yield any result - considering abandoning pBad construct and focussing on the rest.




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{{hide|
{{hide|
*Independent variables :
*Independent variables :
**Temperature range:4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 50<sup>o</sup>
**Temperature range:4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 37<sup>o</sup>C, 45<sup>o</sup>C, 60<sup>o</sup>
**Range may change based upon the initial testing; will only test ranges that in vitro is stable at.  
**Range may change based upon the initial testing; will only test ranges that in vitro is stable at.  
*Dependent variables:
*Dependent variables:

Revision as of 04:06, 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

Introduction

Once the best promoter has been chosen, we would then proceed to characterize it in the in vitro systems. The aim of phase 2 is to define the operating temperature range and life span of the device, and conduct experiments to investigate fluorescence levels with varying temperature changes.

Experiment 1: Degradation Time

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


Materials & Methods

Experiment 1: Degradation Time

Materials Required

  • Reagents
    • 50μl in vitro chassis
    • Known concentration of purified GFP
  • Equipment
    • Fluorometer

Protocol 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.

Constants:

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

Variables:

  • Independent variables :
    • Temperature range:4oC, 15oC, 25oC, 37oC, 45oC, 60o
    • 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)




Experiment 2

Experiment 3

Experiment 4

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