cDNA encoding full length E1FTL➔TVG was created from the wild type E1 template using the QuickChange mutagenesis kit from Qiagen consisting of Pfu polymerase and Dpn1, and cloned into a pET16b expression vector according to the protocol in (Tian 2007 (link)). The E1FTL➔TVG mutant construct employed in this study includes not only the wild type E1 residues 71–73 (FTL) replaced with the corresponding residues of E3 (TVG), but also includes mutation of the single wild type cysteine (residue 106) to serine and an N-terminal MGHHHHHHG- purification tag (139 residues, with tag). The DNA was then transformed into XL1-Blue competent cells and plated on an agar gel plate with appropriate antibiotics and incubated overnight at 37C. Single colonies were picked, used to inoculate LB medium, and mini-prepped using a Qiagen QIAprep Spin kit. The mini-prepped samples were submitted for sequencing to confirm that the mutations were correctly inserted.
Quickchange mutagenesis kit
Manufactured by Qiagen
The QuickChange mutagenesis kit is a tool designed for introducing site-specific mutations in double-stranded plasmid DNA. It enables rapid and efficient mutagenesis without the need for subcloning or the use of specialized E. coli strains.
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Lab products found in correlation
2 protocols using quickchange mutagenesis kit
1
Engineered E1 Mutant Construct
2
Recombinant Expression of S. aureus TarS
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The full-length open reading frame (amino acids 1–573) encoding S. aureus TarS (SAV0258) was cloned into the expression vector pET41b without an affinity tag. Mutagenic TarS constructs were produced with the Quick Change mutagenesis kit (Qiagen). Constructs were transformed into Rosetta (DE3) Escherichia coli. The TarS truncation mutant (TarS1-349) was cloned into the expression vector pET41b with a C-terminal 6x His-tag. Protein expression was carried out overnight at 30°C. Cells were grown in Luria Bertani broth (supplemented with 35 μg/mL kanamycin) to an optical density (600 nm) of 0.6–0.8, at which point Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM. Cells were pelleted and stored at -80°C until required.
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