Mix and go e coli transformation kit

Routine genetic cloning
and plasmid manipulation were carried out in E. coli DH10B cells (New England Biolabs). E. coli ET12567/pUZ8002 was used as the host for intergeneric conjugation
with S. coelicolor as previously described.⁵² E. coli chemically
competent cells were prepared using the Mix and Go! E. coli Transformation Kit (Zymo Research). E. coli was transformed with plasmid DNA via chemically competent heat-shock transformation as described
previously. Plasmid DNA was isolated via the Wizard
Plus SV Minipreps DNA Purification System by following the manufacturer’s
protocols (Promega). All molecular biology reagents and enzymes used
for plasmid construction were purchased from New England Biolabs.
The conjugation donor host E. coli ET12567/pUZ8002
was transformed with constructs for mobilization into S. coelicolor strains, as previously described. For
each transformation, 9–12 independent exconjugants were plated
to DNA plates supplemented with antibiotics and grown for 4–5
days until the formation of vegetative mycelium.

Routine genetic cloning and plasmid
manipulation were carried out in E. coli JM109 (New England Biolabs). E. coli ET12567/pUZ8002 was used as the host for intergeneric conjugation
with S. coelicolor as previously described.^{83 (link)}E. coli chemically
competent cells were prepared using the Mix and Go! E. coli Transformation Kit (Zymo Research). E. coli was transformed with plasmid DNA via
chemically competent heat-shock transformation as described previously.
Plasmid DNA was isolated via the Wizard Plus SV Minipreps DNA Purification
System by following the manufacturer’s protocols (Promega).
All molecular biology reagents and enzymes used for plasmid construction
were purchased from New England Biolabs. The conjugation donor host E. coli ET12567/pUZ8002 was transformed with
constructs for mobilization into S. coelicolor strains, as previously described. For each transformation, 9–12
independent exconjugants were plated to DNA plates supplemented with
antibiotics and grown for 4–5 days until the formation of vegetative
mycelium.

Escherichia coli DH5α (Invitrogen, Schwerte, Germany) was used for cloning, and E. coli BL21 (DE3) (Invitrogen, Schwerte, Germany) was used for protein expression. pET26b (+) vector was purchased from Novagen (Darmstadt, Germany).
Luria-Bertani (LB) media consisted of: 0.5% (w/v) NaCl, 1% (w/v) peptone, and 0.5% (w/v) yeast extract, and sterilized by autoclaving. Agar was added for preparation of standard agar media at 1.5% (w/v). Lysis buffer consisted of: 50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 7.8.
Nco1 and Xho1 restriction enzymes, shrimp alkaline phosphatase, and phusion high-fidelity DNA polymerase were purchased from New England Biolabs (Massachusetts, USA). Trizol^® reagent, T4 DNA ligase, Thermo Scientific RevertAid H Minus first strand cDNA synthesis kit, and PageRuler unstained protein ladder were purchased from Thermo Fisher Scientific (Ohio, USA). Zyppy^TM plasmid miniprep kit, used for plasmid isolation, Zymoclean^TM Gel DNA recovery kit, used for DNA purification, and Mix and Go E. coli transformation kit were purchased from Zymo Research (Irvine, California, USA). HyperLadder^TM 1 kb was purchased from Bioline (London, UK). Protein concentration was determined using Quick Start^TM Bradford protein assay kit from Bio-Rad (Washington, USA).