Introduction

The idea of combining E. coli as an efficient cloning organism since it has the largest library of well characterised parts available and G. xylinus as a robust efficient cellulose producing host came about as a way to take advantage of the characteristics of each host. E. coli DH10B has a very short division time of 30 min whereas the Kombucha Isolated Gluconacetobacter strain has been producing bacterial cellulose robustly in a co-culture with yeast. For these reasons, the aim was to have E. coli produce customisable proteins of interest such as metal binding phytochelatin, linked to cellulose binding domains (CBD), simultaneously with cellulose being produced by G. xylinus allowing these proteins to attach to the material, effectively making it a 1 step functionalisation.

In a co-culture of the cellulose-producing and protein-producing species there will be competition for resources and space, and potentially also a symbiosis of some kind. The main hypothesis of this co-culture experiment is that G. xylinus would orient itself in the oxygen rich layers towards the top of the media due to being an obligate aerobe as explained in the BC synthesis pathway section whereas E. coli would orient itself throughout the media but expectedly have a higher concentration in the oxygen depleted bottom media.

In order to have a stable co-culture, at least in the timescales we are conducting our growth and functionalisation, we need to identify potential interactions and regulate the growth of each species. The main variable identified for the interactions was identified as the carbon source of the HS media used, which requires an initial experiment that can inform the choice/combination of carbon source(s) used for the actual experiment containing E. coli with an Anderson promoter controlled RFP gene (J23104) in J61002 and the Kombucha Isolated strain.

Aims

In addition to the lack of tools, the continuous cellulose production of G. xylinus introduces further problems for genetic engineering, as it results in a low growth rate (the division time of G. xylinus is 4 hours, which is 8 times slower than that of E. coli), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.

Furthermore, although the highest cellulose-producing strain G. xylinus ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.

We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for G. xylinus genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see Functionalisation).

Methods

Results

References