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

Hrp Characterisation: Testing

• Introduction
• Specifications
• Parts
• Devices
• Testing
• Application
• Notes
• References

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

2. Cloning of the Constructs

Link to General Protocol

3. Transformation

Link to General Protocol

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

• Here is a link to an explanation of the Calibration Curves