Ecm 630 electroporator

A codon-optimized gene of wild-type CalB was synthesized for optimal expression in E. coli (GenScript) and cloned into expression vector pET22b+ (MilliporeSigma). A C-terminal 6-histidine Tag was designed to facilitate the purification process of all CalB variants. Using the same codon-optimized WT CalB sequence, three previously reported mutants of CalB displaying increased activity to bulky substrates in nonaqueous conditions were also synthesized: S47L, I189A, and double mutant I189A-L278P^{38 (link),39 (link),46 (link)}. Overexpression of the CalB enzyme from IPTG-inducible vector pET22b+ was tested using 5 different E. coli strains: Rosetta-Gami (DE3), Rosetta (DE3), Rosetta 2 (DE3) PLacI, Origami 2 (DE3), and BL21 (DE3) (MilliporeSigma). The selection markers employed were a combination of kanamycin and chloramphenicol (Rosetta-Gami (DE3)), chloramphenicol (Rosetta (DE3) and Rosetta 2 ((DE3)-PLacI), and streptomycin (Origami 2 (DE3)). Carbenicillin was used as resistance marker for expression vector pET22b+, which was electro-transformed into each strain using 0.1-cm Gene Pulser/MicroPulser electroporation cuvettes (Bio-Rad) and an ECM 630 electroporator (BTX Harvard Apparatus). Cells were recovered in SOB medium⁶² supplemented with 20 µL of 2 M glucose (filter-sterilized) and 2 M magnesium (1 M MgSO₄ and 1 M MgCl₂, autoclave-sterilized).

pKM225 was introduced into stationary phase C. burnetii NMII via electroporation. C. burnetii was made electrocompetent through washing with ice-cold, sterile filtered 10 % (v/v) glycerol prior to electroporation of 50 µl aliquots at 1.8 kV, 500 Ω and 25 µF using 0.1 cm cuvettes and an ECM630 electroporator (BTX Harvard Apparatus). Immediately after electroporation, 950 µl RPMI-glutamax was added to cuvettes. Five hundred microlitres of sample was used to inoculate 6 ml ACCM-2 medium in duplicate. Cultures were incubated overnight, before the addition of chloramphenicol at 3 µg ml⁻¹, followed by a further 4 days of incubation. To obtain colonies, samples were plated onto ACCM-2 agarose plates containing chloramphenicol at 3 µg ml⁻¹. After 14 days of incubation, colonies were washed from the plate and pooled. One hundred microlitres was used to inoculate triplicate 25 ml ACCM-2 cultures for TraDIS library preparation.