Construction of Hib Gene Expression Constructs

A PreScission cleavage site was inserted into the modified p-Mal-c (New England BioLabs) vector pMBP-S3N10-csxA-His₆ (ref. ^{51 (link)}) (tac) resulting in pMBP-PreScission-S3N10-csxA-His₆. The gene cps3B^{27 (link)} was cloned via BamHI/XhoI into plasmid pMBP-PreScission-S3N10-csxA-His₆, replacing the csxA sequence and resulting in pMBP-PreScission-S3N10-cps3B-His₆. The genes bcs1 (OOD27571.1), bcs3 (OOD27573.1; Uniprot: Q2ERG0), rpiA (OOD27170.1), prsA (OOD25971.1) and rk (OOD27132.1) were amplified by PCR using heat-inactivated lysate of Hib strain ATCC 10211 (GenBank accession number MTGI00000000.1) as a template. The resulting PCR products were cloned into pMBP-S3N10-csxA-His₆ via BamHI/XhoI restriction sites or pMBP-PreScission-S3N10-cps3B-His₆ via restriction-free cloning^{52 (link)} replacing csxA and cps3B, respectively. Single amino acid mutants and truncations of Bcs3 were introduced according to ref. ^{53 (link)} or by using the Q5 Site-Directed Mutagenesis Kit (New England BioLabs) according to the manufacturer’s guidelines. Constructs and primers used in this study are listed in Supplementary Tables 4 and 5.

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Cifuente J.O., Schulze J., Bethe A., Di Domenico V., Litschko C., Budde I., Eidenberger L., Thiesler H., Ramón Roth I., Berger M., Claus H., D’Angelo C., Marina A., Gerardy-Schahn R., Schubert M., Guerin M.E, & Fiebig T. (2023). A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule. Nature Chemical Biology, 19(7), 865-877.

Publication 2023

p-Mal-c Q5 Site-Directed Mutagenesis Kit

Amino acid Bcs1 Cleavage Genes Plasmid Primers Site directed mutagenesis Strain Vector

Corresponding Organization : Ikerbasque

Other organizations : BioCruces Health research Institute, Hospital de Cruces, Medizinische Hochschule Hannover, BOKU University, University of Würzburg, CIC bioGUNE, University of Salzburg

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Variable analysis

independent variables

Insertion of PreScission cleavage site into the modified p-Mal-c vector pMBP-S3N10-csxA-His6 resulting in pMBP-PreScission-S3N10-csxA-His6
Cloning of the gene cps3B into plasmid pMBP-PreScission-S3N10-csxA-His6, replacing the csxA sequence and resulting in pMBP-PreScission-S3N10-cps3B-His6
Cloning of the genes bcs1, bcs3, rpiA, prsA, and rk into pMBP-S3N10-csxA-His6 or pMBP-PreScission-S3N10-cps3B-His6, replacing csxA and cps3B respectively
Introduction of single amino acid mutants and truncations of Bcs3

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive control: Not mentioned
Negative control: Not mentioned

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