Engineered Bacillus Strains for Protein Expression

Gene cloning and site directed mutagenesis was done using standard molecular biology procedures as described before^{14 (link), 22 (link)} and B. anthracis Sterne strain genomic DNA. The clones were confirmed with restriction digestion and DNA sequencing (Invitrogen). Site-directed mutagenesis was carried out using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) using GroEL clones as templates. The details of primers and plasmids are provided in Table S1.
Genes encoding for PrkC (1-657 aa), wild-type GroEL (1-544 aa), and GroEL mutants (GroEL-T21A, GroEL-T132A, and GroEL-T329A) were cloned into the E. coli, B. anthracis shuttle vector, pYS5 (modified to express Spectinomycin resistance gene^{63 (link)}), and electroporated in B. anthracis Sterne strain (Bas-wt or BasΔprkC strain) (BTX Electro Cell Manipulator 600).

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Arora G., Sajid A., Virmani R., Singhal A., Kumar C.M., Dhasmana N., Khanna T., Maji A., Misra R., Molle V., Becher D., Gerth U., Mande S.C, & Singh Y. (2017). Ser/Thr protein kinase PrkC-mediated regulation of GroEL is critical for biofilm formation in Bacillus anthracis. NPJ Biofilms and Microbiomes, 3, 7.

Publication 2017

DNA sequencing

Anthracis b Cell Digestion E coli Genes Genomic Plasmids Primers Shuttle vector Site directed mutagenesis Spectinomycin Strain

Corresponding Organization : University of Delhi

Other organizations : Institute of Genomics and Integrative Biology, Savitribai Phule Pune University, National Centre for Cell Science, Laboratory of Pathogens and Host Immunity, Centre National de la Recherche Scientifique, Université de Montpellier, Universität Greifswald

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Variable analysis

independent variables

Site-directed mutagenesis of GroEL (T21A, T132A, T329A)
Expression of PrkC (1-657 aa), wild-type GroEL (1-544 aa), and GroEL mutants in B. anthracis Sterne strain (Bas-wt or BasΔprkC)

dependent variables

Not explicitly mentioned

control variables

Use of standard molecular biology procedures as described before
Use of B. anthracis Sterne strain genomic DNA
Confirmation of clones with restriction digestion and DNA sequencing
Use of QuikChange II XL Site-Directed Mutagenesis Kit
Use of E. coli, B. anthracis shuttle vector pYS5 (modified to express Spectinomycin resistance gene)
Electroporation in B. anthracis Sterne strain (Bas-wt or BasΔprkC)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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