2.1.1 Strains
E. coli K-12 NEB5-α [fhuA2Δ(argF-lacZ)U169 phoA gln V44
Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 ] was obtained
from New England Biolabs (NEB, Ipswich, USA) and used for all cloning
procedures. For recombinant protein expression, linear DNA cartridges
controlled by the T7 promoter were integrated into the bacterial
chromosome at the attTn7 site of E. coli BL21(DE3) [fhuA2
[lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B
int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 ] (NEB). Hereafter, this
strain is referred to as B3<T7-gene >.
Linear DNA cartridges controlled by the A1 promoter were integrated into
the bacterial chromosome at the attTn7 site of E. coli BL21
[fhuA2 [lon] ompT gal [dcm] ΔhsdS] (NEB) containing thelacIQ promoter as described by Glascock and
Weickert.[25] Briefly, the pETAmp-lacIq plasmid
was constructed for integration of the lacIQpromoter in E. coli BL21. This plasmid contains the ampicillin
resistance gene flanked by FRT sites and the lacI gene controlled
by the lacIQ promoter. The pBR322 ori andlacI were amplified from pET30a using the overhang PCR technique
to introduce a C -> T mutation in the lacI promoter.
The linear lacIQ DNA cartridge for genome
integration was amplified using Q5® High-Fidelity DNA
Polymerase (NEB) according to the manufacturer’s manual. Integration
into the bacterial chromosome occurred at the lac operon site ofE. coli BL21 carrying the pSIM5
plasmid.[26] This strain was designated as
BL21Q and referred to hereafter as
BQ <A1-gene >.
The cytosolic protein GFPmut3.1 was used as a recombinant
“easy-to-produce” model protein.[27] An
antigen-binding fragment (Fab) against TNF-α (FTN2) with the DsbA signal
sequence (dFTN2) was used as a recombinant “challenging” model
protein.[28]