Prset emgfp plasmid

In all experiments we used non-motile cells of Escherichia coli top 10 strain [F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG], transformed with pRSET-emGFP plasmid (Thermo Fisher Scientific Corp.) and standard electroporation procedures (Sambrook et al., 1989 ). The plasmid contains T7 promoter regions upstream of the emGFP reporter gene and ApR cassette. Since the cells are deficient in T7 polymerase, the GFP is transcribed only on a basis of the leakage of the promoter leakage. The transformants were cultivated at 37°C on nutrient agar (NA) plates (Sigma-Aldrich) supplemented with ampicillin (100 μg/ml, Sigma-Aldrich)—NAamp.
Prior to the experiments, we prepared overnight liquid cultures from a single colony in NBamp medium. One milliliter of this culture was transferred into 100 mL of the fresh medium and incubated until optical densities appropriate for conducting the particular experiments were obtained. All liquid cultures were incubated with shaking at 37°C and 150 rpm.

The CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to obtain intermediate constructs by direct PCR and restriction/ligation cloning of required genetic elements. The pRSET-EmGFP plasmid (Thermo Fisher Scientific) was used as an expression vector containing the GFP gene. High-fidelity restriction endonucleases BamHI-HF, BglII-HF, EcoRI-HF, NcoI-HF, and PstI-HF; T4-DNA-ligase; T4-polynucleotide-kinase; and shrimp alkaline phosphatase (rSAP) were purchased from New England Biolabs (Ipswich, MA, USA). The oligonucleotides were synthesised by the solid-phase method and purified by preparative polyacrylamide gel electrophoresis (PAGE) by Syntol LLC (Russia). Q5^® High-Fidelity DNA Polymerase (New England Biolabs) was used for all polymerase chain reaction (PCR). Ultrafree-DA Centrifugal Filter Units (Merck, Kenilworth, NJ, USA) were used for DNA extraction from agarose gel. The ZymoPURE™ Plasmid Miniprep Kit (Zymo Research, Irvine, CA, USA) was used for plasmid DNA purification. The authenticity of the plasmids was confirmed by Sanger sequencing performed by Eurogen CJSC (Russia). E. coli strain NiCo21(DE3) (#C2529H, New England Biolabs, Ipswich, MA, USA) was used for cloning and expression experiments according to the manufacturer’s protocol.