Experiments

by UiOslo

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].

Overview of hexosamine biosynthesis pathway and upstream glycolysis in yeast.
Figure 1: Figure 1: Overview of hexosamine biosynthesis pathway and upstream glycolysis in yeast. Reused from a publication by Raimi, et al. (2020) in Royal Society of Chemistry licensed under CC BY 3.0 [9].

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.

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ASSEMBLY

We used New England BioLabs DNA ligation. Protocol largely...

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BRADFORD ASSAY

This is the procedure for Bradford assay.

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CELL LYSIS

This is the procedure for cell lysis.

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CULTURING ESCHERICHIA COLI

Escherichia coli (E. coli) is our intermediate host to proliferate the gene...

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CULTURING KOMAGATAEIBACTER XYLINUS

This is the procedure for culturing K. Xylinus.

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CULTURING SACCHAROMYCES CEREVISIAE

Saccharomyces cerevisiae (S. cerevisiae) is the source of our...

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DNA PRECIPITATION

Nucleotide precipitation is a common technique for concentrating and...

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DPNI DIGESTION

Digestion and restriction cutting enzymes protocol. We use...

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GEL ELECTOPHORESIS AND EXTRACTION

This is the procedure for gel electophoresis and extraction.

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HEAT-SHOCK TRANSFORMATION

This is the procedure for heat-shock transformation.

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HESTRIN-SCHRAMM (HS) MEDIUM

Hestrin-Schramm medium is a common medium composition to...

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LYSOGENY BROTH (LB) MEDIUM

Lysogeny broth (LB) is a nutritionally rich medium which is primarily...

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MINIPREP

We used QIAprep Spin Miniprep Kit. This protocol is based on the official...

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MISCELLANEOUS BUFFERS

This is the procedure for miscellaneous buffers.

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PCR AMPLIFICATION AND PURIFICATION

This is the procedure for PCR amplification and purification.

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PHOSPHATE BUFFERED SALINE

Phosphate-buffered saline (PBS) is a buffer solution (pH ~7.4) commonly...

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DEPHOSPHORYLATION ASSAY

This is the procedure for dephosphorylation assay.

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PREPARATION OF CHEMICALLY COMPETENT E. coli

This is the procedure for preparation of chemically competent E. coli.

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PREPARATION OF CHEMICALLY SUPERCOMPETENT E. coli

This is the procedure for preparation of chemically supercompetent E. coli.

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PREPARATION OF ELECTROCOMPETENT K. XYLINUS

This protocol is a modified version of an existing protocol. To create...

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SDS-PAGE

This is the procedure for SDS-page.

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TRANSFORMATION BUFFERS

Different protocols for producing competent cells has over the years...

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YEAST GENOMIC DNA EXTRACTION

Due to the rigidity of the yeast cell wall, we had to use a...

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YEAST PEPTONE DEXTROSE (YPD) MEDIUM

Yeast peptone dextrose is a medium composition used to grow many common...

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University of Oslo
Digital Life Norway
Evogene
IDT
novozymes
Oslo Mycology Group
Empress Brewery

References

References

  1. Elieh-Ali-Komi, & Hamblin, M. R. (2016)
    Chitin and Chitosan: Production and Application of Versatile Biomedical Nanomaterials
    International Journal of Advanced Research (Indore), 14(3): p.411-427
  2. Lockhart DEA, Stanley M, Raimi OG, Robinson DA, Boldovjakova D, Squair DR, Ferenbach AT, Fang W, van Aalten DMF (2020)
    Targeting a critical step in fungal hexosamine biosynthesis
    J Biol Chem., 295(26): p. 8678-8691
  3. Li S, Kang J, Yu W, Zhou Y, Zhang W, Xin Y, Ma Y (2012)
    Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and essentiality of M. smegmatis MSMEG_1556
    PLoS One, 7(8): e42769
  4. Brown HE, Esher SK, Alspaugh JA (2020)
    Chitin: A "Hidden Figure" in the Fungal Cell Wall
    Curr Top Microbiol Immunol., 425: 83-111
  5. Yadav V, Paniliatis BJ, Shi H, Lee K, Cebe P, Kaplan DL (2010)
    Novel in vivo-degradable cellulose-chitin copolymer from metabolically engineered Gluconacetobacter xylinus
    Appl Environ Microbiol., 76(18): 6257-65
  6. Helenius G, Bäckdahl H, Bodin A, Nannmark U, Gatenholm P, Risberg B (2006)
    In vivo biocompatibility of bacterial cellulose
    J Biomed Mater Res A., 76(2): 431-8
  7. Shirai A, Takahashi M, Kaneko H, Nishimura S, Ogawa M, Nishi N, Tokura S (1994)
    Biosynthesis of a novel polysaccharide by Acetobacter xylinum
    Int J Biol Macromol, 16(6): 297-300
  8. Ogawa R, Miura Y, Tokura S, Koriyama T (1992)
    Susceptibilities of bacterial cellulose containing N-acetylglucosamine residues for cellulolytic and chitinolytic enzymes
    Int J Biol Macromol., 14(6): 343-7
  9. Raimi, Olawale G. and Hurtado-Guerrero, Ramon and Borodkin, Vladimir and Ferenbach, Andrew and Urbaniak, Michael D. and Ferguson, Michael A. J. and van Aalten, Daan M. F. (2020)
    A mechanism-inspired UDP-N-acetylglucosamine pyrophosphorylase inhibitor
    RSC Chem. Biol., 1: p. 13-25
  10. Lee JW, Deng F, Yeomans WG, Allen AL, Gross RA, Kaplan DL (2001)
    Direct incorporation of glucosamine and N-acetylglucosamine into exopolymers by Gluconacetobacter xylinus (=Acetobacter xylinum) ATCC 10245: production of chitosan-cellulose and chitin-cellulose exopolymers
    Appl Environ Microbiol., 67(9): 3970-5