Coding

Part:BBa_K729002

Designed by: Bouran Sohrabi   Group: iGEM12_UCL_London   (2012-06-27)

Laccase for Polyethylene Degradation

Laccase for the degradation of polyethylene and organic pollutants. Sequence pending.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 225
  • 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.

UniversityCollegeLondon Laccase Assay Extracellular.png UniversityCollegeLondon Laccase Activity.png


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 (x10 000) 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) UniversityCollegeLondon LaccaseSEM Water Low mag.png UniversityCollegeLondon LaccaseSEM W3110 Low mag.png UniversityCollegeLondon LaccaseSEM Laccase Low mag.png
High Magnification (10 000x) UniversityCollegeLondon LaccaseSEM Water High mag.png UniversityCollegeLondon LaccaseSEM W3110 High mag.png UniversityCollegeLondon LaccaseSEM Laccase High mag.png

Modelling

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. Combined with the results from the scanning electron microscope, which indicate surface degradation of polyethylene in a relatively short timespan, indicates that this BioBrick holds powerful potential for the break down of polyethylene.

iGEM MITADTBIO_Pune 2019 characterization

Description

Laccase enzyme acts on many different substrates, with its activity also prominent in helping break down polyethylene. Since our aim is to degrade plastic present in menstrual sanitary napkins, it was important to know whether it's activity is affected in the presence of blood. Laccase has mutiple copper ions in its cofactor and therefore it was crucial for us to know whether the activity of laccase is hindered in the presence of other ions present in blood.

Cloning of laccase gene from BBa K729002 into pET28a+

Cloning of laccase in iGEM backbone plasmid will lead to normal low level of laccase expression which will show in vitro activity, but the activity and expressive rate is non-obvious. For the above reasons, we decided to clone laccase gene is pET28a+ expression vector for sustainable production of laccase.
To insert laccase gene into cloning expression vector (pET 28a+), we first designed prefix and suffix primers which contain EcoRI and PstI restriction enzyme sites respectively. The PCR amplification of laccase gene from BBa_K729002 using EcoRI-prefix and PstI-suffix primers give an expected 1.5 kb DNA band as shown in (Fig. 1).

T--MITADTBIO_Pune--ORF.png


Figure 1. PCR amplification of laccase ORF using prefix and suffix primers gave expected size DNA band. M: 500 bp DNA ladder; 1: PCR amplified laccase gene (size 1.5 kb).


The purified PCR amplified products were restriction double digested with EcoRI-PstI, and ligated at same sites in pET28a+ (5.3 kb) expression vector and transformed recombinant plasmid into E. coli (JM109). The transformation result was shown in Figure 2. To check whether the construction was successful, we picked few colonies to extract their plasmids. The inclusion of insert into the recombinant plasmid was verified both by restriction digestion.

T--MITADTBIO_Pune--pet1.png
Figure 2. Restriction digestion of laccase carrying recombinant plasmid using EcoRI. M: 500 bp DNA ladder; 1: Plasmid showing expected size band of 1.55 kb.


We tested the expression of laccase enzyme by transforming the pET28a+ recombinant carrying pET-laccase gene into E. coli expression strain BL21. To overexpress laccase enzyme, E. coli expression strain BL21 containing laccase gene under IPTG-inducible T7 promoter was induced by 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3hrs and 18 hrs at 37°C, corresponding proteins were separated by 12% SDS-PAGE followed by staining with Coomassie Brilliant Blue G250 (CBB). Prior to induction with IPTG, expression of laccase is not detectable by SDS-PAGE (Figure 3 Lane 1). After IPTG induction recombinant, protein bands of about 58 kDa was observed which is in agreement with the theoretical molecular weight of laccase which is 58.87 kDa (Figure 3 Lane 2).

T--MITADTBIO_Pune--SDS.png


Figure 3. Induction and overexpression of recombinant protein: Samples were subjected to 12% SDS-PAGE and stained with Commassie Brilliant Blue G250. A- Lane 1, total cellular proteins from E. coli BL21(DE3) transformed with pET-laccase uninduced; Lane 2, total cellular proteins from E. coli BL21(DE3) transformed with pET-laccase induced by IPTG for 3 hrs; Lane 2, total cellular proteins from E. coli BL21(DE3) transformed with pET-laccase induced by IPTG for 18 hrs.


In vitro laccase assay

We tested whether expressed laccase is active through enzyme activity experiments. To detect laccase enzyme activity, colorimetric assay was carried out using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS method. The nonphenolic dye ABTS is oxidized by laccase into water soluble chromogen ABTS+ which is more stable and preferred state of the cation radical. ABTS+ can be monitored spectrophotometrically at 420 nm. The concentration of the cation radical responsible for the intense blue-green color can be correlated to enzyme activity and is read at 420nm. The supernatant of overnight grown bacteria culture acted as a source of laccase enzyme. The enzyme activity experiment was carried out by using 100 μL of enzyme and 100 μL of ABTS solution (0. 5 mM) in sodium acetate buffer (pH 5). The enzymatic units (U) defined as the amount of enzyme transforming 1 µmol of substrate per minute is calculated by the following formula: Equation : U/L = (∆E×Vt)/(ε×d×Vs)

with ∆E being the change in the extinction of light [min-1] at 420 nm
ε being the molar absorption coefficient of ABTS [M-1 cm-1]
d being the layer thickness [cm] in your cell that the light has to pass
Vt is the total volume measured and Vs is the volume of the enzyme stock solution added to the ABTS stock solution.
It is demonstrated in the given graph by in vitro ABTS assay that recombinant laccase protein can oxidize ABTS. It is essential for our project that recombinant microorganisms should be capable of polyethylene deterioration in the presence of blood and/or dried blood. To check the effect of blood on laccase enzyme activity, we performed the ABTS assay in the presence of different concentrations of blood.

T--MITADTBIO_Pune--plot.png
Figure 4. Laccase activity in presence and absence of blood. The fold differences were calculated compared with control groups and shown in the figure. *P<0.007; **P<0.01; [one-way analysis of variance].

Conclusion

It was clearly observed that the laccase activity is not significantly affected by blood.

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