Cloning and expression of Ku and LigD

Codon-optimized Ku (ID: P9WKD9) and LigD (ID: P9WNV3) sequences from M. tuberculosis were synthesized by Genewiz and cloned through ligation-independent cloning (LIC) (25 (link)) using SspI restriction endonuclease (NEB, R0132L) and pMCSG7 expression vectors with a TEV-cleavable, N-terminal 6xHis-tag (26 (link)) (Addgene). Oligonucleotide primers (Integrated DNA Technologies) are described in Supplementary Table S1. All truncations of Ku and LigD were created by LIC using pMCSG7 or by site-directed mutagenesis (27 (link)). All plasmids were verified using Sanger sequencing (Genewiz).

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Sowa D.J., Warner M.M., Tetenych A., Koechlin L., Balari P., Rascon Perez J.P., Caba C, & Andres S.N. (2022). The Mycobacterium tuberculosis Ku C-terminus is a multi-purpose arm for binding DNA and LigD and stimulating ligation. Nucleic Acids Research, 50(19), 11040-11057.

Publication 2022

Oligonucleotide primers Sanger sequencing

Codon Ligation M tuberculosis Oligonucleotide primers Plasmids Site directed mutagenesis Sspi endonuclease Vectors

Corresponding Organization : McMaster University

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Variable analysis

independent variables

Codon-optimized Ku (ID: P9WKD9) and LigD (ID: P9WNV3) sequences from M. tuberculosis
Truncations of Ku and LigD

dependent variables

Unspecified

control variables

SspI restriction endonuclease (NEB, R0132L)
PMCSG7 expression vectors with a TEV-cleavable, N-terminal 6xHis-tag
Sanger sequencing (Genewiz)

controls

Positive control: Not specified
Negative control: Not specified

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