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Origami 2

Manufactured by Merck Group

The Origami 2 is a compact and versatile lab equipment designed for a range of scientific applications. It features a user-friendly interface and advanced functionalities to assist researchers and scientists in their work. The core function of the Origami 2 is to enable precise and efficient data collection and analysis, supporting the research and development process.

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2 protocols using origami 2

1

Optimized Heterologous Expression of Lipase B

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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-L278P38 (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).
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2

Cultivation and Genetic Manipulation of Bacterial Strains

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Streptococcus pyogenes strain D471 was propagated on solid media in plates containing Todd-Hewitt broth supplemented with yeast extract (0.2%) (THY) and agar (1.4%) or in liquid THY as described by Gera et al [27 ]. S. mutans wild type (Xc) and mutants were grown in Todd-Hewitt broth with 1% yeast extract. All cultures were grown without antibiotics and without aeration at 37°C. E. coli genotypes DH5α (NEB, cat. No. C2988J), DH10α (Thermo Fisher, cat No. 18297010), BL21 (NEB, cat No. C2530H) and Origami 2 (Millipore Sigma, cat. No. 71346) were used for routine plasmid propagation or protein expression and grown in Lysogeny Broth (LB) medium supplemented with either 50 μg/ml kanamycin, 100 μg/ml ampicillin or 35 μg/ml chloramphenicol as needed.
Bacterial strains E. coli CS2775 were transformed with pRGP1 plasmid [28 (link)], gacABCDEFG [23 (link)] to produce pRha or empty plasmid control (pHD0131). The bacterial cells were grown overnight in LB containing erythromycin (150 μg/ml) at 37°C and used next day for whole cell Western blots and FACS and microscopy analysis.
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