Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1) The GenBank accession number for the DNA sequence reported in this paper is AF125322.

Marolda, Cristina L.; Feldman, Mario F.; Valvano, Miguel A.

doi:10.1099/00221287-145-9-2485

Cited by 56 publications

(54 citation statements)

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“…[22] The two modified S-layer protein forms A_SgsE_T12 and G_SgsE_T12 with the PelB signal peptide, the extended N-glycosylation site from C. jejuni replacing the O-glycosylation site T 620 and a C-terminal hexahistidine-tag were cloned from pET22b into pBAD24 vector, which is arabinose inducible. The gene cluster necessary for the synthesis of E. coli O7 antigen provided through plasmid pJHCV32 [24] was introduced in E. coli CLM24 together with PglB and A_SgsE_T12 or G_SgsE_T12. After induction of protein expression with arabinose over night, the SgsE neoglycoprotein was analyzed by Western immunoblotting using anti-SgsE-antibody.…”

Section: Transfer Of E Coli O7 Antigen To the Engineered N-glycosylamentioning

confidence: 99%

“…Plasmids pACYC184pgl, [21] pJHCV32, [24] pEC(AcrA-per), [28] and pMAF10 [22] have been described previously. All strains and plasmids are listed in Table 1.…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 99%

“…coli CLM24 cells containing the plasmids pMAF10 (encoding the oligosaccharyl/protein transferase PglB from C. jejuni, [22] pJHCV32 (encoding E. coli O7 antigen [24] ), and pBAD24-PelB_A_SgsE_T12 or pBAD24-PelB_G_SgsE_T12 (encoding periplasmic SgsE forms), were induced by the addition of arabinose to 0.02% (wt vol −1 ). After induction at 37 °C for 5 h, arabinose was added again to ensure protein expression when the carbon source becomes limiting (as arabinose can be metabolized by the cells).…”

Section: Production Of An Sgse Neoglycoprotein With E Coli O7 Glycosmentioning

confidence: 99%

“…[11] Recent demonstrations of the functional transfer of protein glycosylation pathways into the experimental model organism Escherichia coli at the genetic level opens new avenues for carbohydrate engineering of S-layer proteins. [21,22] Based on the availability of molecular tools, the Pgl protein N-glycosylation system from Campylobacter jejuni [23] and the E. coli O7 antigen biosynthesis system [24] were used for the design of SgsE-neoglycoproteins. The C. jejuni Pgl enzymes synthesize a heptasaccharide with the structure D-GalNAc-α1,4-D-GalNAc-α1,4-(D-Glc-β1,3)-D-GalNAc-α1,4-D-GalNAc-α1,4-D-GalNAc-α1,3-D-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-D-Glc, [25] involving the lipid carrier undecaprenylpyrophosphate.…”

Section: Introductionmentioning

confidence: 99%

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Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Steiner

Hanreich

Kainz

et al. 2008

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Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S-layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self-assembly protein as a cellular prerequisite for Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts protein glycosylation, and on engineering of one of the natural protein O-glycosylation sites into a target for N-glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co-or post-translationally transferred to the S-layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the Slayer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S-layer protein self-assembly system. Tailor-made ("functional") nanopatterned, self-assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics.

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Section: Transfer Of E Coli O7 Antigen To the Engineered N-glycosylamentioning

confidence: 99%

“…Plasmids pACYC184pgl, [21] pJHCV32, [24] pEC(AcrA-per), [28] and pMAF10 [22] have been described previously. All strains and plasmids are listed in Table 1.…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 99%

Section: Production Of An Sgse Neoglycoprotein With E Coli O7 Glycosmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Steiner

Hanreich

Kainz

et al. 2008

Small

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“…The product of this reaction serves as an acceptor for the addition of subsequent sugars to complete the biosynthesis of O antigen or enterobacterial common antigen repeating subunits. In the case of the O7 polysaccharide, a model system used in our laboratory, the repeating subunit consists of a backbone of GlcNAc, galactose, mannose and Nacetylviosamine, and a side chain of rhamnose (L'Vov et al, 1984 ;Marolda et al, 1999). Once complete, the O7 subunit is translocated to the periplasmic surface of the inner membrane by a process that requires the Wzx protein .…”

Section: Abbreviationsmentioning

confidence: 99%

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

Amer¹,

Valvano²

2001

Microbiology

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A model‐based development approach for the verification of real‐time Java code

Pour

Strooper

Wellings

2011

Concurrency and Computation

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Many real-time systems are safety-and security-critical systems and, as a result, tools and techniques for verifying them are extremely important. Simulation and testing such systems can be exceedingly timeconsuming and these techniques provide only probabilistic measures of correctness. There are a number of model-checking tools for real-time systems. Although they provide formal verification for models, we still need to implement these models. To increase the confidence in real-time programs written in real-time Java, this paper proposes a model-based approach to the development of such programs. First, models can be mechanically verified, to check whether they satisfy particular properties, by using current real-time model-checking tools. Then, programs can be derived from the model by following a systematic approach. We introduce a timed automata to RTSJ Tool (TART), a prototype tool to automatically generate real-time Java code from the model. Finally, we show the applicability of our approach by means of four examples: a gear controller, an audio/video protocol, a producer/consumer and the Fischer protocol. N. HAKIMI POUR, P. STROOPER AND A. WELLINGS semantics. The RTSJ provides a type-safe object-oriented programming language, like standard Java. However, it refines some of the semantics for existing Java classes in order to provide more predictable execution times.A real-time model is a simplified representation of a real-time system. Models focus on system behaviour and abstract many details of programs [14]. More importantly, these models can be verified mechanically with real-time model checkers. The contributions of this paper are: introducing a model-based approach to increase the confidence in the correctness of real-time programs written in RTSJ and presenting tool support for this approach. Timed automata [15] are used as the modelling language, since timed automata have well-defined mathematical properties and a simple graphical representation. Moreover, timed automata can capture both qualitative and quantitative features of real-time systems [16]. The next step is to mechanically verify the model using the UPPAAL model checker [6]. UPPAAL has a graphical user interface; it is well-used and wellsupported. After verifying the model, a mapping between the model features and RTSJ is used to derive the RTSJ code from the model. The mapping guarantees that certain properties, such as safety and bounded liveness properties, that hold in the model also hold in the generated code, although timing invariants in the model need to be checked separately. We also introduce our prototype tool to generate RTSJ programs from UPPAAL models.The next section reviews theories and languages that have been proposed for modelling realtime systems and the related work on real-time model checking. An overview of our approach is provided in Section 3. The details of the mapping are presented in Section 4. We introduce the timed automata to RTSJ Tool (TART) in Section 5. Section 6 presents the application of the approach ...

show abstract

Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1) The GenBank accession number for the DNA sequence reported in this paper is AF125322.

Cited by 56 publications

References 54 publications

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

A model‐based development approach for the verification of real‐time Java code

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