Escherichia coli DH5-α
(New England Biolabs) was used as the cloning and testing strain in
this work. Escherichia coli BL21 was used as the
protein expression strain for downstream protein purification. Cells
were grown in LB-Lennox (10 g/L casein peptone (Oxoid, ThermoFisher
Scientific), 5 g/L yeast extract (Oxoid, ThermoFisher Scientific),
and 5 g/L NaCl (VWR) supplemented with 15 g/L agar (Oxoid, ThermoFisher
Scientific) for agar plates) supplemented with the appropriate antibiotics
(Sigma-Aldrich). All enzymes were purchased from New England Biolabs.
Primers were ordered from Eurofins Genomics or Sigma-Aldrich (primer
list:Table S1 ). PCR reactions were performed
with Q5 polymerase (NEB) unless specified otherwise. Colony PCR reactions
were performed with the Taq polymerase (NEB). A QiaQuick
PCR purification kit (Qiagen) and a QIAprep plasmid Miniprep kit (Qiagen)
were used for the purification of PCR products and plasmid DNA, respectively.
DNA sequences were confirmed by Sanger sequencing performed by Eurofins
Genomics.
(New England Biolabs) was used as the cloning and testing strain in
this work. Escherichia coli BL21 was used as the
protein expression strain for downstream protein purification. Cells
were grown in LB-Lennox (10 g/L casein peptone (Oxoid, ThermoFisher
Scientific), 5 g/L yeast extract (Oxoid, ThermoFisher Scientific),
and 5 g/L NaCl (VWR) supplemented with 15 g/L agar (Oxoid, ThermoFisher
Scientific) for agar plates) supplemented with the appropriate antibiotics
(Sigma-Aldrich). All enzymes were purchased from New England Biolabs.
Primers were ordered from Eurofins Genomics or Sigma-Aldrich (primer
list:
with Q5 polymerase (NEB) unless specified otherwise. Colony PCR reactions
were performed with the Taq polymerase (NEB). A QiaQuick
PCR purification kit (Qiagen) and a QIAprep plasmid Miniprep kit (Qiagen)
were used for the purification of PCR products and plasmid DNA, respectively.
DNA sequences were confirmed by Sanger sequencing performed by Eurofins
Genomics.