VII. Aqua di Cutinase



This group project won the first prize for the Audience Choice Award at the BIOMOD competition organized by Wyss Institute for Biologically Inspired Engineering at Harvard University.

overview.
Aqua di Cutinase a.k.a. microplastic free water.


background.
Since the late 19th century plastic has been an integral part of our daily lives. By virtue of this polymer-family our world has experienced a bright upswing, from car-tyres and bags to calculators and shampoos. It's world wide production and use had a good reason, since it has an enormous amount of properties and can be processed easily. Unfortunately, millions of tons of plastic waste are produced each year, which is detrimental for our planet and with that, the health of all organisms living on it.

The awareness of the plastic problem has been increased over the past few years. However, society is far for from solving this problem, which seems to worsen every minute. Over the last five decades, global plastic consumption rose from 5 to 100 million tons per year, most of which is completely unnecessary and a waste of valuable resources. The fact that as many as 600 billion plastic bags are being redundantly produced each year or that only 1% out of the produced 14 million tons of Styrofoam is recycled annually pose a tremendous problem. Via ingestion by plankton and fish it enters the food chain and can end up in human food. In fact, our whole natural environment suffers from plastic.


Plastics can be divided into macro (> 5mm in diameter) and microplastics (≤ 5mm in diameter). Macroplastics can be found in plastic bags, bottles, car materials, etc. Microplastics, on the other hand, are present in various everyday products, like peelings, creams and toothpaste. Or can be found in the drain when washing your fleece jacket or microfiber towel. Even the potable water flowing out of our taps contains microplastics. Unfortunately, there is not much awareness or knowledge about how harmful they are to humans.



Research is already underway to develop new techniques to convert macroplastics into fuels in an environmentally friendly way. For removing microplastics, me and the team at Biokatalyse TU Berlin aimed to construct a modular filter which can be applied to potable water production in order to break down microplastics into less harmful compounds which in turn are absorbed by charcoal leaving the polluted water clear from microplastics and chemicals.


scientific approach.
The goal was to produce a nano-membrane carrying an immobilized flagella forest which is also biodegradable with the aid of synthetic biology. For that we needed to design, functionalize and produce three components:

💧the nano-membrane
💧the root flagellin
💧the fusion proteins

💧The nano-membrane
We synthesized a cellulose acetate membrane with silica linkers and alkyne groups. The linkers performed a click reaction with the non-canonical amino acid L-azidohomoalanine (AHA), forming a benzene ring and therefore a stable bond with the protein containing AHA.

💧The root flagellin
To increase the catalytical surface for the enzyme, we performed an in vitro reaggregation of flagellin monomers by Flagellin-self-assembly.
The flagellin consists of four linearly connected domains labelled D0, D1, D2 and D3, which are arranged from the inside to outside of the flagellar filament (Vonderviszt and Namba, 2010-2013).




To attach the root flagellin onto the cellulose acetate membrane, we modified the D0 domain by substituting the start methionine by AHA and replacing the second amino acid lysine by glycine, what prevents the cleavage of the start methionine and permits the alkyne group to click to the AHA group of the flagellin, making the root flagellin work as an anchor for the entire flagella filaments.

💧The fusion proteins
In addition, we suppressed the D3 domain of flagellin in the genetic design and added short GS-linker segments to the N- and C-terminus of the genes for Cutinase or GFP. We designed two fusion proteins. Flagellin-GFP, for visualizing the flagellum structure as well as controlling whether the root flagellin clicks to the membrane using fluorescence microscopy. And flagellin-cutinase. Cutinase is the enzyme used for the polyethylene terephtalate (PET) degradation in our water. It is an α/β-hydrolase enzyme that hydrolyzes water-soluble esters and insoluble triglycerides (Walton et al.,1972). PET is degraded into ethylene glycol and terephtalic acid (Sulaiman et al, 2012). The fusion protein flagellin-cutinase offers us the possibility to produce PET degrading flagella.

The protein genes were inserted into the pET30b vector which allowed us to make the following constructs:



We tested the protein production for the best expression conditions.


The resulting construct resembles a flagella forest, growing on a cellulose membrane, anchored by the root flagellin, with leaves and branches formed by fusion flagellins. It offers a huge catalytic surface that lights up fluorescent green in case of flagellin-GFP fusion proteins and degrades PET in case of flagellin-cutinase fusion proteins.

Fluorescence microscopy was performed to analyze whether the GFP_AHA is properly clicked on the membrane and to analyze if the assembly of the flagellins as a fusion protein works.



SEM images were obtained to analyze the porosity of the membrane, thereby, comparing the functionalized membrane without any clicked proteins and the membrane with clicked GFP.

A pNPB assay was carried out to compare the efficiency of our purified wildtype Cutinase to a commercially purchased Esterase.



© Juliette van Haren 2019    ︎  ︎