Gene ruler 1 kb marker

Enzymes for DNA restriction and for cloning were obtained from New England Biolabs (Ipswich, MA) or Thermo Fischer Scientific (Schwerte, Germany). The Gene JET plasmid miniprep kit, PCR purification kit, and Gene Ruler 1-kb marker were from Thermo Fisher Scientific and Qiagen (Venlo, Netherlands). N-Acetylmuramic acid (MurNAc) was from Bachem (Bubendorf, Switzerland), and the DNA dye NonTox was from Applichem (Darmstadt, Germany). Oligonucleotides were purchased from MWG Eurofins (Ebersberg, Germany) and are listed in Table S1 in the supplemental material.

B. subtilis 168 glpQ was amplified by PCR with the primers pET28a-glpQ-for and pET28a-glpQ-rv (MWG Eurofins, Ebersberg, Germany). Oligonucleotide primers are listed in Table S1. The PCR products were purified (Gene JET Purification Kit and Gene Ruler, 1-kb marker, Thermo Fisher Scientific), digested with the appropriate restriction enzymes (New England Biolabs), and ligated with T4 DNA ligase (Thermo Fisher Scientific) into the expression vector pET28a (Novagen), allowing them to overproduce a C-terminal His₆ tag fusion protein. E. coli BL21 (DE3) cells carrying pET28a-glpQ were grown as described above and lysed in a French pressure cell. The His-tagged GlpQ protein was purified by Ni²⁺ affinity chromatography using a 1-ml HisTrap column (GE Healthcare), followed by size exclusion chromatography on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare), and purity was checked with 12% SDS-PAGE. The purity of the enzyme was confirmed via SDS-PAGE (Fig. 2A). From a 1-liter culture, 3.6 mg of GlpQ was obtained. The enzyme was stored at a concentration of 0.23 mg/ml at −20 °C in 0.1 m Tris-HCl buffer (pH 8).