Composite

Part:BBa_K5246042

Designed by: Edgaras Zaboras   Group: iGEM24_Vilnius-Lithuania   (2024-09-25)
Revision as of 14:43, 29 September 2024 by Sarpilo (Talk | contribs) (→‎Experimental characterization)


C.Crescentus CB2/CB2A hfsH-hfsK-hfsL Part of the polysaccharide tetrad assembly system

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 is a part of the complete holdfast tetrad assembly system BBa_K5246043 used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/. This part can also be used separately for glycolipids consisting of glucose, mannosaminuronic acid, and N-acetyl-D-glucosamine synthesis, but this feature needs more detailed characterization.

Usage and 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 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 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] -->Full functional project operon assembly with this part and full operon characterization can be found in composite part BBa_K5246043.

Usage and Biology

TBA

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 96
    Illegal XbaI site found at 856
    Illegal PstI site found at 945
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 96
    Illegal PstI site found at 945
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 96
    Illegal BglII site found at 859
    Illegal XhoI site found at 1674
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 96
    Illegal XbaI site found at 856
    Illegal PstI site found at 945
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 96
    Illegal XbaI site found at 856
    Illegal PstI site found at 945
    Illegal NgoMIV site found at 154
    Illegal NgoMIV site found at 178
    Illegal NgoMIV site found at 182
    Illegal NgoMIV site found at 255
    Illegal NgoMIV site found at 381
    Illegal NgoMIV site found at 542
    Illegal NgoMIV site found at 2223
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

Experimental characterization

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

===References===

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