G.xylinus

Overview

Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for Gluconacetobacter genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with G.xylinus genetic engineering, and turned G.xylinus KI and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for G.xylinus genetic engineering.

Key Achievements

• Isolated a new strain of Gluconacetobacter (named G. xylinus igem) from Kombucha tea and characterized its properties fully.
• Sequenced the previously unknown genomes of G. xylinus ATCC 53582 and G. xylinus igem strains - the first genomes sequenced in the history of iGEM
• Discovered four new plasmids capable of replication in Gluconacetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in G. xylinus ATCC 53582 and igem strains as well as in E. coli
• Were the first in science to create transgenic cells of G.xylinus igem strain
• Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for G. xylinus engineering and expressed them in the ATCC 53582 and igem
• Developed a set of new and improved protocols for efficient genetic engineering of G. xylinus
• In summary, turned G. xylinus ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters

E. coli

Overview

Escherichia coli is the organism of choice for synthetic biology. It is thoroughly characterised, easy to engineer and has numerous parts, control circuits and complex constructs tested in this host.

Some E. coli strains have evolved to produce low amounts of cellulose but the machinery under specific regulatory control (its secretion is generally linked to biofilm formation and stress situations). In order to expand the use of bacterial cellulose as a functional biomaterial that is cost effective to produce, it is desirable for its production machinery to be implemented in an organisms that is easy to engineer and has proven success in large scale bioreactors.

Here, we confirm that the high output cellulose production machinery of Gluconacetobacter Xylinus can be transferred into other organisms. We have proven function of the Acs cellulose producing operon in Escherichia coli using Congo Red binding assays.

Key Achievements

• Contributed an optimised Asc cellulose production operon to Registry of Standard Parts.
• Proved the portability of Gluconacetobacter xylinus operon.
• Assembled a fully synthetic, functional, cellulose-producing system in Escherichia coli
• Demonstrated the synthesis operon in a two plasmid system for separate induction of genes

RFP Co-Culture

Overview

Based on the hypothesis of E. coli BL21D3 operating anaerobically and G. xylinus replicating aerobically, a co-culture experiment for bacterial cellulose was attempted. Initially, the best possible carbon source among 5 available was found experimentally. This data was used to inform the choice of glycerol and glucose as the carbon feedstock. With these carbon feedstocks, E. coli with RFP expressed were successfully embedded in the bacterial cellulose, and RFP was clearly visible.

Key Achievements

• Experimentally determined the optimum carbon feedstocks for a HS media based co-culture of E. coli and G. xylinus.

Functionalisation

Overview

Attaching functional proteins to cellulose can expand the properties of cellulose and allow us to selectivity capture specific contaminants in water. By creating fusion proteins of sfGFP and metal-binding proteins with five different cellulose binding domains, three of which are new to the registry, we were able to characterise the relative affinities of the CBDs and show proof-of-concept removal of contaminants from water.

Key Achievements

• Added three new cellulose binding domains to the registry
• Performed an assay with sfGFP-fusions to determine the strongest potential binders which could be carried forward for further testing
• Identified potential metal binding proteins to bind heavy metals from wastewater, including the addition of a new promising polypeptide phytochelatin EC20
• Tested metal ion capture ability of the CBD-phytochelatin fusion constructs when attached to cellulose

Mechanical Testing

Overview

To check the feasibility of using our bacterial cellulose as a customisable ultrafiltration membrane, the mechanical properties were tested. For this to work, we checked the tensile strength of our filter, calculated the shear stress and estimated the processing method that produces membrane with the longest life-time. Our ultimate tensile stress calculated matched with literature values of bacterial cellulose within standard deviation, and the data analysis provided the insight to use non-blended cellulose rather than blended..

Key Achievements

• Introduced a new aspect to the manufacturing track in terms of quantitatively characterising the mechanical properties of our product, bacterial cellulose
• Quantified the tensile stress-strain properties of our bacterial cellulose.
• Discovered that our bacterial cellulose can sustain an order of magnitude higher water pressures than those typically used for ultrafiltration membranes.
• Established that non-blended compared to blended bacterial cellulose was significantly more ductile, and would have longer life time as a water filter, which informed the choice of cellulose for the final product.
• Tested and analysed 20 samples of bacterial cellulose test pieces.
• Created a size and quality optimised gif for visualisation of the experiment and written a protocol for future iGEM teams to do the same.

Water filtration

Overview

By attaching functional proteins to cellulose we can expand it's properties and selectivity capture specific contaminants in water. We used five different cellulose binding domains and fused them to different metal binding proteins, and sfGFP. We performed assays to test the binding of the CBD fusions to our cellulose.