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