Team:Athens/Poster

MORPHÆ

An engineered biofilm for the production of a novel structurally coloured material

Presented by iGEM Athens 2020*

D. Aslanis, K. Belegri, G. Broutzakis, A.A.T. Dekmak, M.I. Ioannidou, S. Kanellopoulos, E. Kelefioti Stratidaki, K. Pylarinou, N.E. Salpea, V. Spyridaki, I. Toumpe, O.Z. Veloudiou


*National and Kapodistrian University of Athens, National Technical University of Athens, Agricultural University of Athens

Project Description

Colours in nature can be the result either of chemical pigments or of the physical structure of surfaces. Certain bacterial strains exhibit structural colour when they form biofilms, a phenomenon caused by spatial geometries in the micron scale. In this iGEM project we utilize bacteria from the Flavobacterium genus that display structural colour naturally, to create a material that is coloured due to that property. In order for ​Flavobacteriia to secrete a cellulose based extracellular matrix that retains this property, genes from the bcs operon of Komagataeibacter xylinus will be transferred. A biophysical mass-spring model of the cell will be developed to simulate the gliding motility mechanism based on the mechanical interactions between the cells, along with a simulation predicting the optical properties of a known structure. A kinetic modelling for the cellulose biosynthesis will also be implemented to better predict the final structure of the biomaterial.

The problem of synthetic colour

Toxicity of chemical compounds
The main compounds involved in the production of synthetic colour are indicated to have harmful effects to humans and other living organisms.

Environmentally harmful production
The textile industry accounts for 20% of industrial water pollution, while the dyeing process and the treatment of textiles demand high consumption of water and energy.

Intensive Labour
Poor working conditions for the production of paints often lead to working accidents and harmful effects on human health.

Idea

We genetically manipulate bacteria from the Flavobacterium genus in order to produce cellulose and release it extracellularly. These bacteria naturally produce structural colour, due to their spatial arrangements, so if this structure is retained by the cellulose produced, the end-product will also appear coloured. Therefore, by isolating the extracellular matrix from the bacteria, we will obtain an acellular coloured biomaterial.

BioBricks

Ιn order for the Flavobacteriia to produce cellulose, appropriate genes are inserted into the plasmid. The genes from the bcs operon of Komagataeibacter xylinous bacteria were selected. Each gene will be flanked with the appropriate suffixes and prefixes, as to connect with the ompA promoter, Flavobacteriia specific RBS and Rho independent terminator using the Golden Gate Type IIS assembly standard, creating a transcriptional unit for each one. Each transcriptional unit will also be flanked with the appropriate suffixes and prefixes in order to connect both with one another and with the plasmid backbone, forming Level 1 Biobricks and following again the Type IIS Assembly. Similarly, the upstream genes from Komagataeibacter xylinous will also be incorporated into the plasmid between the inverted repeats of the Himar1 transposon using the Golden Gate Type IIS assembly standard. The transcriptional units then will be transferred inside the genome with the Himar1 transposon through random distribution of insertions.

Experimental Procedure

The insertion of the plasmid is done with conjugation using the Escherichia coli S17-1 strain as a donor. Selection of this process is done by using two different antibiotic resistance genes (KanR and ErmR). The first is used for the selection of the recombinant E. coli and the ErmR gene for the selection of the Flavobacteriia since it is expressed in them but not on E. coli.

Modelling of gliding motility

Flavobacterium johnsoniae cells translocate themselves along their long axis without the aid of pili or flagella by a mechanism defined as gliding motility. This type of motility is indicated to contribute to pattern formation and thus structural colour appearance.

Theoretical Mathematical model in MATLAB
We extended a previous theoretical mathematical model that can predict and explain their movement and interactive forces by adding an extra rotational force to the bacteria. It is a mass-spring model that was simulated using MATLAB.

Video 1. (a) The torque of the bacteria is zero. As expected, the result is similar to the one obtained by the original model. (left) (b) The torque changes the direction of the bacteria after their collision significantly. (right)

Simulation in Box2D

We created a simulation based on Newtonian interactions using Box2D, a 2D physics library. We examined the case of head-to-side collision between two cells moving in perpendicular directions and then we scaled it up to a large population of bacteria moving in different random directions.

Video 2. (a) Simulation of two flexible cells. We assumed strong attachments between the cells and the substrate. The primary cell maintains its direction after the collision and the secondary cell aligns with the former. (top left) (b) Without the cell-substrate force, both cells changed directions and aligned to a new common one after the collision. (top right) (c) Simulation of 500 flexible cells. (bottom left) (d) Simulation of 500 rigid cells. (bottom right)

Simulation of the optical properties

A key goal of our project was to predict the colouration of the proposed biomaterial. Using the Finite Element Method (FEM), Maxwell’s Equations were solved on a simplified geometry based on observations of TEM cross-cut images of our system.

Reflection spectra of the agar-bacteria system.

Reflection spectra of the cellulose-bacteria system.

Reflection spectra of the acellular biomaterial.

The results are able to approach experimental data, and predict that a cellulose-bacteria system will have structural colouration. Lastly, an acellular biomaterial based on cellulose does not seem to retain the structural colour of the colonies, however further optimization must be done to finalize the expected outcome.

Kinetic modelling of cellulose production

A kinetic modelling of cellulose production is essential to predict the time period needed to create the desired material. This model integrates mRNA and protein expression alongside bacterial growth kinetics to better describe cellulose production.

Once wet lab data is acquired for our system, parameter optimization will be implemented to provide better estimations about the kinetics of cellulose production.

Integrated Human Practices

efficiency
Utilisation of Flavobacteriia biofilm and simulation of its optical properties.

safety
Selection of cellulose as the main biomaterial component.

ethics
Transparency of the production process behind the end-product.

universality
Popularising the production methods and organising further optimisation of the end-product to meet society’s needs.

sustainability
Efficiency, safety, universality and ethics subsumed under the umbrella of sustainability.

Science Communication

School of Ursulines
Introducing students to the world of SynBio.

Café Scientifique Athens
Communicating SynBio and MORPHÆ to a broad audience.

Athens School of Fine Arts
Showcasing structural colouration to the Greek BioArt community.

Future goals

What to expect from MORPHÆ

• Execute the designed Wet lab experiments


• Evaluate & Optimize our Dry Lab outcomes


• Design a detailed Scaling Up Process


• Build and incorporate Hardware to accommodate our Wet Lab experiments


• Conduct a Sustainability Reporting


• Further develop the Greek 3D BioArt community

Acknowledgements

Attributions

Sponsors & Supporters

Dr. Panagoula Kollia
Dr. Evangelos Topakas
Dr. Vasiliki Koumandou
Dr. Colin Ingham
Dr. Michalis Kavousanakis
Elena Pappa
Special thanks to iGEM Athens 2018 and iGEM Athens 2019

REFERENCES

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