Difference between revisions of "Part:BBa K5246025"

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CB2/CB2A HfsG Glycosyltransferase, 6xHis tag for purification

Introduction

Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - C. crescentus and H. Baltica - harness 12 protein synthesis pathways to produce sugars anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used E. coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.

This protein is part of the Tetrad assembly system BBa_K5246043 and operon responsible for addition of N-acetyl-D-glucosamine to N-acetyl-D-glucosamine and deacetylation of the said molecule BBa_K5246041.

Part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/.

This part also has a non 6xhis-tagged variant BBa_K5246007.

Biology and usage

Biology

Caulobacter crescentus is a common freshwater gram-negative oligotrophic bacterium of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, C. crescentus daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure called holdfast, allowing them to adhere to surfaces and perform cell division.[1][2]

Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface[3][4].

The C. crescentus holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in Escherichia coli.

The holdfast synthesis (hfs) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway[4][5][6].

HfsG, in particular, is responsible for transfer of N-acetyl-D-glucosamine to a sugar acceptor.

HfsG

HfsG gene encodes a cytoplasmic protein of 309 aa homologous to family 2 glycosyltransferases. Transferrs sugar units from UDP-GlcNAc to oligosaccharide, catalyzes the polymerization of GlcNAc. C.Crescentus HfsG mutants were completely devoid of holdfast material

Usage

Proteins of the holdfast synthesis system assemble a short chain of sugar monomers in a specific sequence on a lipid carrier - a glycolipid.

Glycolipids are predominantly located on the extracellular surface of eukaryotic cell membranes and are responsible for various functions such as receptors for viruses and other pathogens, allowing them to enter a specific host cell that has unique glycolipid markers. This feature can let us use said glycolipids as labels for a precise and targeted liposome distribution throughout the body, delivering anything from cancer drugs to gene editing systems directly to the target cells.

To create a liposome labeling system, we had to select specific proteins that could be utilized for this purpose. Following bioinformatics analysis using the Conserved Domain Database, Protein BLAST, DeepTMHMM, and AlphaFold 3, we identified five proteins of interest from each strain: HfsG, HfsH, HfsJ, HfsK, and HfsL.

To utilize these enzymes, it was essential to develop a suitable purification strategy. For efficient cloning, we chose Golden Gate assembly. For efficient purification, we selected immobilized ion affinity chromatography (IMAC) as our purification method, based on recommendations from one of the few available papers where C. crescentus proteins were expressed and purified from E. coli. We opted for conventional 6x histidine tags (his-tag) to facilitate straightforward purification. It was crucial to determine the appropriate terminus for 6xHis-tag insertion to avoid disrupting the protein conformation and lessening purification efficiency.

Sequence and Features

Experimental characterization

References