AqualoseHaroon Chughtai, Michael Florea, Laura de Arroyo Garcia, Deze Kong, Christopher Lazenbatt, Christopher Micklem, Despoina Paschou, Gabriella Santosa, Xenia Spencer-Milnes

Customisable Ultrafiltration Membranes from Bacterial Cellulose

References:1. Chen, P., Kim, H.-S., Kwon, S.-M., Yun, Y. S. & Jin, H.-J. (2009) Current Applied

Physics. 9(2, Suppl. 1): 96-992. Oshima, T., Kondo, K., Ohto, K., Inoue, K. and Baba, Y. (2008) Reactive and

Functional Polym 68, 376–383.

3. Lee, K. Y., Buldum, G., Mantalaris, A., & Bismarck, A. (2014) Macromolecular bioscience, 14(1), 10-32.

4. Zhang, Mengmeng; Wang, Bin; Xu, Bingqian J. (2014) Phys. Chem. B, 118, 6714−6720s

The Water Problem

A Bacterial Cellulose Filter

Attributions

A growing worldwide population and increasing development makes water shortages increasingly severe

Bacterial cellulose is pure and strong with nanometre-scale pore size1,2. It naturally absorbs common contaminants, including heavy metals2. Due to these properties, we used it as basis for our UF membrane.

Special thanks to:Benjamin Reeve, Henrik Hagemann, Catherine Ainsworth, Nicolas Kral, Kirsten Jensen, Dr Tom Ellis, Prof. Paul Freemont, Prof. Richard Kitney, Dr Geoff Baldwin, Dr Guy-Bart Stan

Biosafety

Water is a global issue – by 2030, more than 50% of the world’s population are expected to suffer from water stress. We have created a new type of water filtration membrane from bacterial cellulose, which can help solve this problem. By adding proteins which bind specific contaminants and increasing cellulose production in G. xylinus and E. coli, we made a next generation filter that is inexpensive, hyper-modular and adaptable.

Discussing with over 20 charities and companies, and compiling a water report, we identified that water recycling and ultrafiltration (UF) were the limiting technologies

Cost Effective Manufacture Material Properties

Expanded and Adaptable

Increasing G. xylinus Productivity

G. xylinus - A New Chassis

Our manufactured membrane is free of living cells after pasteurisation and so is certified as non-GM for sale

Bacterial cellulose is produced by Gluconacetobacter xylinus3 whose native cellulose operon consists of acsABCD

Our project sought to address this through manufacture of a better UF membrane using the synthetic biology design cycle

Functionalising Bacterial Cellulose Modelling CBD Binding

To ease engineering of new cellulosic biomaterials, we created a G. xylinus toolkit consisting of 4 new plasmids and 40 parts

Introduction

We transformed G. xylinus with VHb haemoglobin (K1321200), increasing biomass production 2-fold (N=3, error=SD)

Transferring Production Into E. coliWe made E. coli produce cellulose by refactoring a high-producing cellulose operon (acsAB + acsCD) & transforming on 2 separate plasmids (K1321336 + K1321335)

Genome map of our isolated G.xylinus igem strain Created a new functionalised cellulosic UF membrane

Created a library of cellulose-binding fusion proteins

Achieved cellulose production in E. coli

Sequenced two genomes

Made a G. xylinus toolkit consisting of over 40 parts

Created over 100 constructs

Mass produced bacterial cellulose

Collaborated with London BioHackspace & many others

Imperial iGEM team in a Thames Water purification plant

In order to meet our specification we: ✓ Engineered E. coli and G. xylinus to increase cellulose yields and reduce manufacturing costs

✓ Bound proteins to cellulose to remove specific contaminants

✓ Determined how to process, mass-produce and incorporate the filter into existing technologies

Cost AnalysisComponent Media

Quantity (l)

Source Quantity Price per unit ($)

Water, sugar, green tea, vinegar

4 Off the shelf, London

4 l 0.90

Bacterial cellulose

4 60 cm x 40 cm tray

0.24 m2 14.9

In order to assess scalability of bacterial cellulose we bulk produced & harvested >70 pellicles for further processing

In our treatment we found that cellulose held dye well & could be processed into many textures, shapes & forms

The cost of materials using our mass manufacture protocol

is significantly lower than existing alternatives

and makes it commercially

viable

Scaling Production

Processing Cellulose

Cost Analysis

Collaborated with artists to use our

Including single cell analysis

Using parameters from wet-lab assays we created an ODE model to predict the saturation rate of binding sites as a function of initial CBD concentration4

We used cellulose-binding domains

(CBDs) as basis for attaching functional proteins to cellulose including phytochelatins for binding metals and sfGFP for assaying CBDs

Our System Works!

Our membrane modularly slots into existing filtration systems improving their contaminant targeting abilities

i in iGEM Cellulose production was verified using a Congo Red assay

Nickel concentration in filtrate between filters

Nic

kel c

once

ntra

tion

(ppb

) [Ni+] decreased from 32000 ppm

to 1.5 ppm

We used our phytochelatin-dCBD bacterial cellulose membrane to filter nickel ions proving that our membrane is better than raw cellulose

Explored the

Art & Design

Interlab Study

And...

Frequency of Country in iGEM Top 6Achievements

language and international character of the competition

cellulose as a textile for fashion

Increasing productivity via Vhb expression

OD

600

90% CBS saturation against initial CBD concentration

Tim

e fo

r 90

% C

BS s

atur

atio

n (s

)

Initial concentration of CBD in solution (M)

G. xylinus CFUs after treatment

Colo

ny fo

rmin

g un

its

Our expressed CBD-sfGFP fusion protein

Stress-strain characteristics of BC

Tens

ile S

tres

s (M

pa)

Strain (%)

High KO model shows standard G. xylinus

grows at surface where oxygen is plentiful

Low KO model shows VHb strain, that grows over a wider range and so increases BC yields

California in 2011 (above) and 2014 (below)

Sustainable solutions are required to mitigate the effects of water stress

We also sequenced the genomes of two G. xylinus strains including our kombucha isolated “igem” strain

An industrial plate and frame, dead end filtration setup where our membrane could be used

UF membranes operate under high pressure so we tensile tested our manufactured biomaterial to failure to validate our approach

Mechanical Testing

We successfully processed our bulk produced cellulose into a filter like material which we functionalised with our CBD fusions