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Part:BBa_K322125:Experience

Designed by: Chris French and Richard Partridge Hicks   Group: iGEM10_Edinburgh   (2010-10-08)

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Applications of BBa_K322125

Ed10-RedLightSensCharData2.jpg


lacZ RLS Characterisation

Red light = less yellow colour ONPG
No Red light = more yellow colour ONPG

Further characterisation of the RLS was attempted using the Beta-Galactosidase Assay (A Better Miller) from OpenWetWare (http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller)) and preliminary data was recorded and calculated as shown above. The cultures grown were as follows:

  • JM109 – RLS.lacZ.YFP (Light / Dark)
  • envZ – RLS.lacZ.YFP (Light / Dark)
  • JM109 – lacZ Control (Light / Dark)
  • JM109 – RLS.lacZ (Light / Dark)

and the conditions were Cml 40 & IPTG 90 in similar ONs to those used in the YFP characterisation.

This characterisation data was acquired using a β-Galactosidase substrate o-nitrophenyl-β-D-galactoside (ONPG) which is cleaved to form a yellow dye that can be easily visualised and absorbance measurements can be taken. This can be used as a direct measure of lacZ activity as lacZ encodes for β-Galactosidase and when ONPG is in excess the production of the visible yellow dye can be measured over time to shown lacZ activation and control via promoter systems.

The absorbance at 420nm was divided by the time taken for the colour of o-nitrophenyl to become visible and was used to calculate a rate of expression.

This preliminary Miller-like assay shows limited useful characterisation date of the Red Light Sensor (RLS) system, and is a good start towards accurate characterisation of the system, which we hope to continue now that a preliminary methodology has been found and tested.

The envZ – RLS.lacZ.YFP (Light / Dark) cultures did not produce Miller-like results, as was expected and have such not been included in the graphs, they were however run as a negative control to prove that any effects seen were not due to an artefact from the envZ mutant. Though the data is not entirely useful we believe it may be due to a number of potential causes – such as the cofactor not being expressed properly, weak light intensity unable to activate the RLS system, or insufficient expression of the system as a whole - all of which we could test for given sufficient time, allowing us to improve our experimental technique to account for accordingly. We will endeavour to do so and bring further results to the Jamboree.


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