Part:BBa_K5246045
C.Crescentus CB2/CB2A hfsF-hfsC-hfsI Part of polysaccharide export apparatus
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 polymerization and export apparatus BBa_K5246046 used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/. This part can also be used separately for polysaccharide export, 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_K5246046
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 2813
Illegal PstI site found at 1121
Illegal PstI site found at 2620 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1121
Illegal PstI site found at 2620
Illegal NotI site found at 2293 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2039
Illegal BglII site found at 2411
Illegal BglII site found at 2816
Illegal BglII site found at 3872
Illegal BamHI site found at 181
Illegal XhoI site found at 3286 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 2813
Illegal PstI site found at 1121
Illegal PstI site found at 2620 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 2813
Illegal PstI site found at 1121
Illegal PstI site found at 2620
Illegal NgoMIV site found at 431
Illegal NgoMIV site found at 539
Illegal NgoMIV site found at 821
Illegal NgoMIV site found at 878
Illegal NgoMIV site found at 922
Illegal NgoMIV site found at 953
Illegal NgoMIV site found at 1177
Illegal AgeI site found at 239
Illegal AgeI site found at 3066 - 1000COMPATIBLE WITH RFC[1000]
Experimental characterization
Part cloning
All of the proteins composing this system are responsible for polysaccharide polymerization and export. Since the system's proteins are found in the membrane, we concluded that using a low-copy plasmid would decrease the probability of inclusion body formation. Their formation would diminish the functionality of our system, as the proteins would not allow the polysaccharide to be exported outside the bacteria.
To assemble specifically this part into BBa_K5246046 to then further assemble the holdfast synthesis pathway in E. coli , we had to assemble this part first into a backbone of pACYC-Duet-1 containing other composite part genes: BBa_K5246044. We designed a strategy to maximize the success of plasmid assembly by first assembling plasmids with 3 genes (part:BBa_K5246044) and, after verifying the sequences, integrating 3 left genes into that backbone (Fig. 1). In this way, we prevented Golden Gate assembly errors by trying to construct plasmids from 8 or more fragments.
The BBa_K52460465 assembly was done using Golden Gate assembly with IIS AarI restriction enzyme sites introduced during PCR amplification. The backbone of pACYC-Duet-1 (Novagen) and fragments were amplified using Phusion Plus DNA polymerase, as the genome of C. crescentus has a high GC% content making the appearance of non-specific products during PCR amplification more common and primer design more challenging (Fig. 2). Since hfsA gene had an AarI RE site directly in the gene, this site was domesticated during side directed mutagenesis.
Due to the high amount of non-specific products, the fragments were gel-purified. Vectors and fragments composing this operon were mixed in equimolar amounts with GG reaction components and incubated as described in the protocol. The reaction was later transformed into E. coli Mach1 (Thermo Scientific) competent cells. The assembly was then confirmed with restriction digest analysis (Fig. 3), and positive colonies were sequenced.
After acquiring sequence-verified clones, we further cloned the 3 remaining genes: hfsF,hfsC, hfsI, this was done the same way as the first 3 genes. After transformation into E. coli , obtained colonies were once again screened with cPCR (Fig. 4) and restriction digestion analysis (Fig. 5). After choosing positive colonies full plasmids with hfsA-hfsB-hfsD-hfsF-hfsC-hfsI were once again sequenced and fully characterized as a composite part BBa_K5246046.
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
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