Part:BBa_K729006

J23119-RBS-Laccase

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 286
  
• 1000
  COMPATIBLE 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.

The Scanning Electron Microscope (SEM) images below show the effects of subjecting Low Density Polyethylene (LDPE) to three different sets of conditions in order to visualise the degradative properties of cells containing our Laccase construct (Bba_K729006). We can then also compare this with the ability of untransformed E.coli W3110 and mechanical shear in water to breakdown the LDPE target. All samples were in suspension at 37°C and 200rpm in an incubated shaker for 3 days. Low magnification (x100) images were used to observe the macro-trend in zonal enzymatic activity. There appears to be increased ‘roughness’ (shown by the small protrusions on the surface as well as areas of darker shading, which represent areas of greater contour), across the surface of the plastic as we progress from the sample treated with water to untransformed E.coli W3110 to E.coli transformed with the UCL Laccase construct. High magnification (x10k) images were taken to give more high resolution information regarding the activity of the enzyme on a much smaller scale. The SEM images highlight increased amounts of ‘pitting’ (the generation of small holes in the plastic due to it’s structural breakdown), as we go from water to E.coli W3110 to bacteria containing Bba_K729006. These pits are shown by the much darker areas. This small scale breakdown has a cumulative effect which results in the overall trend seen at the lower magnification. In conclusion, this collection of SEM images indicates an ability both of the Bba_K729006 transformed cells to produce the Laccase enzyme at greater titres than untransformed bacteria (though the E.coli W3110 is still seen to have some degradative effect), and of the Laccase produced to degrade the polyethylene target.

SEM Results

	Water	W3110	Laccase
Low Magnification (100x)
High Magnification (10 000x)

Conclusion

Through the use of a simple quantitative assay, it has been ascertained that E. coli, transformed with our construct has a significantly increased extracellular laccase activity when compared with a control cell line. This indicates that this BioBrick holds powerful potential for the break down of polyethylene.