Experiments

Research

During our design process, we figured out a problem we wanted to solve. From this we found a paper from Yadav, et al. (2011) [1]. This paper is a proposal for how to make a copolymer of cellulose and chitin in situ using cellulose producing bacteria cloned with genes from yeast. We choose to use Saccharomyces cerevisiae instead of Candida albicans as in the original research, and we wanted to expand the scope of production of this material.

To produce chitin monomers, its precursor UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a nucleotide sugar. N-acetylglucosamine (GlcNAc) is the monomer forming chitin and UDP-GlcNAc is the precursor to chitin. Chitin is the second most abundant polysaccharide after cellulose [1], a structurally similar polysaccharide. UDP-GlcNAc is synthesized by the hexosamine biosynthesis pathway (HBP), a pathway consistent of conserved enzymes, albeit mediated through different intermediate compounds in prokaryotes and eukaryotes, found in most living organisms [2, 3]. Despite the enzymes existing in most bacteria, taking part in synthesis of peptidoglycan, the expression is significantly lower than in yeasts, which depend on this pathway to produce chitin, a component of its cell wall [4].

The primary motivation to upregulate the expression of hexosamine biosynthesis enzymes is to produce a more biocompatible polymer then cellulose alone with specific applications in medicine and/or applications where high crystalline cellulose has a disadvantage as outlined more clearly by Yadav, et al and Helenius, et al [5, 6]. It has been proven that Komagataeibacter can incorporate amino-sugars in its endogenous cellulose synthase, creating a lysozyme-susceptible copolymer [7, 8].

Figure 1 is an overview of the hexosamine pathway. There are two entry points for substrate to enter biosynthesis UDP-GlcNAc. The first entry point is diffusion of a Glucosamine (GlcN) across the cell membrane from the extracellular space. This entry of UDP-GlcNAc biosynthesis by extracellular Glucosamine has been studied with limited success [7, 10]. This in turn limits the incorporation of non glucose (Glc) monomers by cellulose synthesis in Komagataeibacter as hypothesized and demonstrated by Yadav et al. [5]. They focused on this route of entry by expression of relevant enzymes from Candida albicans and observed an 18-fold increase in GlcNAc vs Glc monomers in their modified cellulose with the presence of extracellular GlcN. The other entry of substrate into UDP-GlcNAc biosynthesis is directly from glycolysis. Fructose-6-phosphate enters biosynthesis of UDP-GlcNAc by four different enzymes: GFA1, GNA1, PCM1/AGM1 and QRI1/UAP1. This is the pathway that we are modifying to improve the incorporation of GlcNAc vs Glc monomers by synthesis of UDP-GlcNAc from glucose directly. To increase the concentration of intracellular UDP-GlcNAc, genes related to hexosamine biosynthesis GFA1, GNA1, PCM1/AGM1 and QRI1/UAP1 are transformed into the mutant from Saccharomyces cerevisiae.

Experiments

The source of our genetic material used as recombinant DNA is Saccharomyces cerevisiae. To extract the DNA, we designed primers and amplified the target genes from DNA extracted using lithium acetate to lyse the yeast. The DNA is further purified by gel extraction to a solution containing ideally only one gene. This material was then used in the transformation of our bacteria.

We have used the plasmid backbone pASG-IBA3 (IBA3), pUC19 and pBBR to clone our DNA sequences into our hosts. It was kindly provided to us by the Evogen laboratory. All plasmids are used in some way in our project, but to different purposes. IBA3 was as part of troubleshooting, because of bad experiences with pUC19 to assemble the DNA into out intermediate host Escherichia coli. This was necessary, as we didn’t know if it was the genetic construct or the design/method of insert that lead to poor results of cloning. pBBR is the plasmid used to insert the final construct from the intermediate host to transform Komagataeibacter xylinus. All plasmids are selected for in antibiotics containing medium, ampicillin for IBA3 and pUC19, and kanamycin for pBBR. Different transformation techniques were used in E. coli and K. xylinus, based on best practise found in literature. E. coli was transformed by preparation of competent cells in either calcium chloride or Inoue buffer and is transformed with heat-shock treatment. K. xylinus is prepared competent in HEPES buffer and transformed with electroporation.

When the bacteria were transformed, and growth on selective agarose plates were observed, the colonies were both suspended in a colony PCR and inoculated as a liquid culture in selective medium. From this, PCR of vector content using primers and gel electrophoresis is used to check if the insert is roughly the correct size. If this is the case, the liquid culture will be used for miniprep and lysis. The miniprep product is used for sequencing and lysate is used for protein analysis. The protein analysis consists of SDS-page to determine if protein is expressed by size, while a dephosphorylation assay is used to determine increased activity versus the parental strain.

Protocols

Below you can read and download all the protocols we used for our project work.