Difference between revisions of "Part:BBa K729002"
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| − | Our results indicate a significantly higher rate of oxidation | + | Our results indicate a significantly higher rate of oxidation from the cells containing our BioBrick than the control cell line. This indicates that our transformed ''E. Coli'' have successfully produced laccase, and in significant enough quantities that it is released into the extracellular space. This allows it to oxidise the syringaldazine utilised in the laccase assay. |
[[Image:UniversityCollegeLondon_Laccase_Assay_Extracellular.png|600px]] | [[Image:UniversityCollegeLondon_Laccase_Assay_Extracellular.png|600px]] | ||
Revision as of 14:31, 21 September 2012
Laccase for Polyethylene Degradation
Laccase for the degradation of polyethylene and organic pollutants. Sequence pending.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 225
- 1000COMPATIBLE WITH RFC[1000]
Description
The laccase enzyme has been shown to degrade polyethylene when being expressed by a number of different bacteria and fungi. This is due to conserved copper binding sites which couple the oxidation of a substrate with the cleavage of dioxygen bonds, leading to the capability to degrade plastics (particularly polyethylene).
By driving the laccase production using a strong constitutive promoter, we expect overexpression of the protein which will allow it to be secreted into the extracellular medium where it can act upon the target plastic.
Characterisation
Plastic degradation is mediated via a laccase protein. As such, we will be using an enzymatic activity assay to determine that the laccase enzyme is expressed. Laccase catalyses the oxidation of syringaldazine, a reaction that exhibits an observable OD change at 530nm. A sample of syringaldazine can be used as a blank in a spectrophotometer, against a sample containing syringaldazine and our laccase sample, allowing the rate of oxidation to be measured, and hence the enzymatic activity of laccase.
In order to determine the effectiveness of laccase in degrading plastic, we will expose strips of various types of plastic to the laccase expressing bacteria, before viewing the strips under a scanning electron microscope. This will allow us to compare the pitting in plastic samples treated with laccase, to the untreated samples, allowing us to determine the extent of plastic degradation.
Results
Our results indicate a significantly higher rate of oxidation from the cells containing our BioBrick than the control cell line. This indicates that our transformed E. Coli have successfully produced laccase, and in significant enough quantities that it is released into the extracellular space. This allows it to oxidise the syringaldazine utilised in the laccase assay.
