Difference between revisions of "Part:BBa K5246030"

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===Experimental characterization===
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===Protein expression===
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<h2><i>Hirschia baltica</i></h2>
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<p>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.</p>
  
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====Bioinformatic analysis====
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<p>After SDS-PAGE gel analysis, we concluded that we successfully expressed HfsG, HfsH, HfsK, and HfsL proteins from <i>H. baltica</i>.</p>
  
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CDD and protein BLAST analysis suggest that HfsG is a glucosyltransferase family 2 protein. Proteins of this family are involved in cell wall biosynthesis. HfsG is similar to the WecA protein in E.Coli that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate. In the case of HfsG, it catalyzes the transfer of UDP-GlcNAc to the sugar acceptor made earlier in the holdfast synthesis pathway.
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<p><b>HfsG</b> is visible on the left side of the gel (Fig. 1).</p>
  
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Protein topology analysis using DeepTMHMM suggests that HfsG is a globular protein located in the cytoplasm. AlphaFold 3 structures further confirm it. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions.
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      <h3>Table 1. <i>H. baltica</i> protein sizes in kDa</h3>
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        <tr>
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          <th>Protein Name</th>
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          <th>Size (kDa)</th>
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        </tr>
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        <tr>
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          <td>HfsG</td>
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          <td>37</td>
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        </tr>
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        <tr>
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          <td>HfsH</td>
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          <td>29</td>
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        </tr>
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        <tr>
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          <td>HfsJ</td>
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          <td>41</td>
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        </tr>
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        <tr>
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          <td>HfsK</td>
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          <td>28</td>
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        </tr>
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        <tr>
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          <td>HfsL</td>
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          <td>36</td>
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        </tr>
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      </table>
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HfsG is family 2 glycosyltransferase similar to WecA of E.Coli. This globular protein transfers UDP-GlcNAc to the acceptor molecule, our conclusions are in agreement with existing research. [1][2][3]
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        <figcaption><center><b>Fig. 1.</b> 12% SDS-PAGE analysis of <i>H. baltica</i> in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific). </center></figcaption>
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===References===
 
===References===

Latest revision as of 19:30, 1 October 2024


HB 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 the addition of N-acetyl-D-glucosamine and deacetylation of the former molecule BBa_K5246042.

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_K5246024.

Biology and usage

Biology

Hirschia baltica is a common marine of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, H. baltica 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 a 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 H. baltica 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][9].

HfsG in particular is responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule.

HfsG

GThe HfsG gene encodes a 331 amino acid protein, homologous to family 2 glycosyltransferases, in Hirschia baltica. This protein transfers sugar units from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to the growing chain in the synthesis of the holdfast polysaccharide repeat units in the cytoplasm. Studies have shown that HfsG mutants were completely devoid of any 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 852
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 747
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Protein expression

Hirschia baltica

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 HfsG, HfsH, HfsK, and HfsL proteins from H. baltica.

HfsG is visible on the left side of the gel (Fig. 1).

Table 1. H. baltica protein sizes in kDa

Protein Name Size (kDa)
HfsG 37
HfsH 29
HfsJ 41
HfsK 28
HfsL 36
Fig. 1. 12% SDS-PAGE analysis of H. baltica in BL21(DE3) before expression and after induction at 0.5 mM IPTG concentrations for 3 hours at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

References

1. Hendrickson, H., & Lawrence, J. G. (2000). Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. FEMS Microbiology Reviews, 24(2), 177–183. https://doi.org/10.1111/j.1574-6976.2000.tb00539.x
2. Andrews, S. C., Robinson, A. K., & Rodríguez-Quiñones, F. (2004). Bacterial iron homeostasis. Journal of Bacteriology, 186(5), 1438–1447. https://doi.org/10.1128/jb.186.5.1438-1447.2004
3.Rabah, A., & Hanchi, S. (2023). Experimental and modeling study of the rheological and thermophysical properties of molybdenum disulfide-based nanofluids. Journal of Molecular Liquids, 384, 123335. https://doi.org/10.1016/j.molliq.2023.123335
4. Boutte, C. C., & Crosson, S. (2009). Bacterial lifestyle shapes stringent response activation. Journal of Bacteriology, 191(9), 2904-2912. https://doi.org/10.1128/jb.01003-08
5. Mackie, J., Liu, Y. C., & DiBartolo, G. (2019). The C-terminal region of the Caulobacter crescentus CtrA protein inhibits stalk synthesis during the G1-to-S transition. mBio, 10(2), e02273-18. https://doi.org/10.1128/mbio.02273-18
6.Thanbichler, M., & Shapiro, L. (2003). MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Journal of Bacteriology, 185(4), 1432-1442. https://doi.org/10.1128/jb.185.4.1432-1442.2003
7. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.
8. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.
9. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.