Team:Hong Kong HKUST/Description

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• Project
  Overview PE Degradation Alkane Metabolism Alkane Results MFC Design MFC Result Experimental protocol Notebook Parts
• Modelling
  Laccase Alkane Metabolism Alkane and MFC (FBA)
• Human Practices
  Education and Engagement Collaborations Integrated Human Practices
• InterLab
• Medal Requirements
• Safety
• Team
  Team Members Attributions

From plastics to the power line

Polyethylene is the most widely used plastic and arguably one of the most versatile materials to ever be synthesized. Its practicality and convenience however, have come at a great environmental cost. Polyethylene takes millennia to decompose, leaching harmful microplastics into the environment. We approached this pressing issue from a synthetic biology perspective, making use of E. coli engineered with genes encoding for laccase to degrade polyethylene into smaller alkane chains. Our team recognizes the opportunity to further advance this project by addressing another key issue – energy. Using Shewanella oneidensis MR-1 strain’s inbuilt extracellular electron transport mechanism in tandem with genes responsible for alkane metabolism derived from Desulfatibacillum alkenivorans, we will generate electricity from the metabolism of degraded polyethylene, hoping that it will one day help in solving the world’s growing energy needs. Thus, our project serves as an integrated effort to simultaneously solve two crucial problems.

To achieve our goal of electricity generation from plastic degradation, we separated our project into three independent but interconnected modules. The first stage, the PE degradation module, focused on creating a genetic construct that would readily synthesize and secrete Laccase in order to degrade the polyethylene into smaller alkanes. Once fragmented, the alkanes would be processed by our second stage - the alkane metabolism module. This module plans to introduce alkane uptake and metabolism pathways into Shewanella oneidensis MR-1 so that it can process alkanes and channel the electrons obtained from its cellular respiration. The third and final module was the physical construct of the microbial fuel cell. We housed the Shewanella within a microbial fuel cell of our design to characterise and optimise the electricity generated. The following is a schematic to describe the flow of the project.

Figure 1. Protein structure and enzymatically active sites of laccase ^[7].

 

Laccase belongs to a family of enzymes known as multicopper oxidases which oxidize a variety of substrates while reducing dioxygen to water in the process. As shown in Figure 1, The enzymatically active part of laccase involves a cluster of 4 copper ions at different oxidation states ^[4]. The catalytic mechanism begins with the transfer of electron from substrate to the T1 copper site as a result of its higher redox potential; the electron obtained from the reduced T1 copper is then passed through the intermediate electron acceptor, T3 copper site and eventually ends up at the T2 copper site ^[4]. The T3 copper serves as an electron acceptor in the aerobic oxidation process while the presence of the T2 copper site is necessary as a terminal oxygen reducing site. Laccases were long reported to be present in some lignin- bio-degrading fungi, where they catalyse the oxidation, including carbonyl formation, of aromatic compounds. Nonetheless, there is considerable evidence showing laccase’s affinity to non-aromatic substrates, such as saturated hydrocarbons.

 

Due to the lack of any hydrolysable functional group, a specific enzymatic attack on the PE backbone may not be favourable; however, a random oxidation on PE could effectively weaken its chemical integrity. Presently, the activity of laccase on PE has been confirmed by documented cases from scientific research papers as well as from past iGEM projects. For instance, University London College IGEM team 2012 proposed that the laccase could lead to increased deterioration of polyethylene structure and was confirmed by the SEM (Scanning Electron Microscope) which showed an evident scratch made on a PE film surface after incubation with laccase, compared to the control. Moreover, in research conducted by other teams, crude laccase secreted by rhodococcus ruber was incubated with LDPE of average molecular weight 191,000. It reportedly resulted in 15 to 20 percent of mass reduction over a period of two weeks, which demonstrated the feasibility of laccase-degradation of polyethylene. To further enhance the efficiency of laccase, introduction of copper ions and mediators to the reaction medium were also justified to yield a higher degradation rate ^{([3], [5])} .

 

The target of this module was to demonstrate the ability of Laccase, expressed by E.coli, to fragment PE into simple hydrocarbons. For the purpose of utilizing laccase in the outer-membrane space, outer membrane protein A (OmpA)  is required to serve as a protein signal to the membrane so that the laccase can be dumped to the intracellular space. Meanwhile, to minimise the misfolding of laccase with OmpA, our chosen OmpA includes a linker sequence at the N-termini to separate laccase from the signalling protein OmpA. To aid in protein extraction for characterization, a 6X his-tag region is added to all of our laccase. The final effective construct for activity assay is as follows:

 

 

Apart from the construct above, another construct was also involved to characterize the effectiveness of OmpA in bringing out laccase to the intracellular space.

With these two constructs constitutively expressed in E.coli, it is expected that we can compare the differences of laccase quantity in the culturing medium of E.coli for our characterisation.