IGEM:IMPERIAL/2007/Projects/Hrp System/Testing

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Hrp Characterisation: Testing



Proposed Schedule

Motivation

  • The motvation behind the characterisations carried out is to create well characterised parts and devices, and to characterise in terms of; GFP molecules synthesized cfu -1 sec-1 .

Dependencies

General tasks to be completed for each of our constructs:

  1. Amplifying the biobricks
  2. Cloning of the constructs
  3. Transformation
  4. Growing Cultures
  5. Fluorometer
    1. Measuring Inputs - GFP downstream of Promoters
    2. Measuring Outputs- GFP downstream of Devices
  6. Calibration

General Protocols

1. Amplifying the biobricks

2. Cloning of the Constructs

Link to General Protocol

3. Transformation

Link to General Protocol

4. Growing Cultures

5. Fluorometer

  • The fluorometer used in these experiments is 'The Twinkle'.
  • This machine is a plate reader fluorometer that can measure 96 well plates.
  • Using this machine it is hoped that absorbance at 600nm and the fluorescence emission of acGFP can be measured at set time intervals.
  • The machine is being installed 3rd of August 10.30am.


  • The general principle is to measure the fluorescence into the device and out of the device, this allows characteristics such as transfer function to be calculated.
  • To enable us to measure inputs and output, two separate experiments will be carried out, one for the inputs fluorescence and one for the output of fluorescence.
  • A Hrp Protocol can be found here. This is a general protocol and can be applied to the various devices and parts to be characterised.
  • Two specific fluorescence measurements will be made for each device:
  1. Input -The input of fluorescence will be measured at varying inducer concentrations, to give the value of GFP molecules synthesized cfu -1 sec -1 into our device at specific inducer concentrations.
  2. Output -The output of fluorescence will be measured at varying inducer concentrations and at various time intervals. The inducer concentration will have a known GFP molecules synthesized cfu -1 sec -1 input and so we will be measuring the time response and level of output of our device.

6. Calibration

  • The two measurements that will be made for the various devices are fluorescence emission and absorbance.
  • The aim of the characterisation is to use these two measurements in order to create a more generic unit to allow for modular design and easier repetition of experiments. The unit that has been chosen to be used is GFP molecules synthesised cfu -1 sec-1 .
  • In order to convert our raw data into these units, two sets of calibration curves are needed.
  1. Absorbance
  2. Fluorescence
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