Part:BBa_K196002
HfsG protein from Caulobacter crescentus
Caulobacter crescentus is an aquatic, Gram-negative bacterium that divides asymmetrically. Besides the bacterium is able to synthesize a strong glue. This glue is mainly composed of polysaccharide. Different proteins are required in the holdfast synthesis, export and attachment in C.crescentus (for more details, please refer to the "introduction" section). In our project, we wanted to implement the glue production in Escherichia coli. As E. coli possesses homolog proteins, we decid to insert the hfsG and hfsH genes in a plasmid. HfsG is a glycosyltransferase and HfsH [1] is a carbohydrate esterase. Here is the BB for the hfsG gene.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 480
Illegal AgeI site found at 379
Illegal AgeI site found at 864 - 1000COMPATIBLE WITH RFC[1000]
Contribution by Vilnius-Lithuania iGEM 2024
During the project a native gene variant was used, amplified via PCR directly from the genome of C. crescentus CB2 and CB2A strains. The amino acid sequence is identical to the BBa K196002 part, with a his tag added in the N terminus.
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
Protein expression
CB2 strain
We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.
After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.
HfsG is visible on the left side of the gel (Fig. 2).
Table 1. C. crescentus protein sizes in kDa
| Protein Name | Size (kDa) |
|---|---|
| HfsG | 34 |
| HfsH | 27.9 |
| HfsJ | 34.7 |
| HfsK | 43.3 |
| HfsL | 33.3 |
CB2A strain
We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.
After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.
HfsG is visible in the center of the gel (Fig. 2).
Table 2. C. crescentus protein sizes in kDa
| Protein Name | Size (kDa) |
|---|---|
| HfsG | 34 |
| HfsH | 27.9 |
| HfsJ | 34.7 |
| HfsK | 43.3 |
| HfsL | 33.3 |
Protein purification
CB2 strain
After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.
HfsG HfsG was purified and clearly seen in the elution fraction of the gel.
CB2A strain
After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.
HfsG HfsG was purified and clearly seen in the elution fraction of the gel.
| None |