Luria broth lb

Fecal samples were sent for analysis at Statens Serum Institut, Copenhagen, Denmark. Ten μg fresh stool sample was mixed in 2 mL phosphate buffered saline (PBS, pH 7.38) and 10 μL was plated on SSI enteric medium agar plates (SSI, Hillerød, Denmark, product no. 724) and incubated overnight at 37 °C. Bacteria from the SSI enteric medium were harvested and plated on SSI blue agar plates (SSI, Hillerød, Denmark, product no. 694) selective for gram negative bacteria and incubated overnight at 37 °C. E. coli colonies were isolated from the SSI blue agar plate and tested for Beta-glucuronidase40 ((PGUA), SSI, Hillerød, Denmark, product no. 1033) and Indol (Biomérieux, Denmark, product no. 56541). Isolated E. coli were inoculated in Luria broth (LB) (Sigma-Aldrich, GmbH, Germany) and incubated overnight at 37 °C. Twenty-five μL of bacterial culture in LB were diluted in 975 μL of sterile water, boiled at 100 °C for 15 min, centrifuged for 10 minutes at 14,000× g, and the supernatant (bacterial DNA) was transferred to a new tube and stored at −20 °C for PCR testing. E. coli isolates with different colony morphologies from the SSI blue agar plates were harvested, dissolved in 15% glycerol (Sigma-Aldrich, GmbH, Germany. Cat: 67757) in beef stock (SSI, Hillerød, Denmark, product no. 1056), and stored at −80 °C.

The N-terminal coding region of MAG2 (aa 284 to 407) was PCR amplified using Q5 High-Fidelity DNA polymerase (New England Biolabs) and MAG2 N-terminal specific primers and ligated into pET32 vector. The resulting vector was transformed into BL21(DE3) competent Escherichia coli (NEB). Transformed bacteria were grown in Luria broth (LB; Sigma-Aldrich) containing ampicillin (100 μg/μl) for 5 to 6 h at 37°C and were induced with isopropyl β-d-1-thiogalactopyranoside (0.1 mM) for protein expression. The bacterial cells were pelleted, resuspended, and sonicated in lysis buffer (1% Triton X-100–10 mM imidazole–7 mM beta-mercaptoethanol–PBS), and the resulting supernatant was purified with nickel beads. Purified peptides were emulsified with Titer Max Gold and injected intraperitoneally into BALB/c^Δdm1 mice. Immunized mice were boosted every 14 days before sera were collected 1 month after initial immunization and checked for reactivity against in vitroT. gondii cysts by IFA. All primers used are listed in Table 2.

Restriction enzymes were purchased from New England Biolabs (NEB). All polymerase chain reactions, unless mentioned otherwise, were performed using Q5 High fidelity DNA polymerase (NEB). PCR primers were designed using GeneRunner program Version 6.5.52¹ Primers used in the study were custom synthesized from Integrated DNA Technologies. Various ingredients to reconstitute minimal media (M9) or Luria broth (LB) were purchased from Sigma or VWR.

Representative bacteria of the genus Escherichia-Shigella were selected to be included in live bacteria vaccine formulations as previously reported (17 (link), 18 (link)). Anti-microbiota vaccinations were used to test the impact of host immune response against targeted bacteria on mosquito microbiota composition and structure, mosquito survival and Plasmodium infection. The Escherichia coli strains BL21 (DE3, Invitrogen, Carlsbad, CA, USA), and O86:B7 (ATCC^® 12701TM) were selected. The two bacterial strains were prepared as previously described (17 (link), 18 (link)). Briefly, E. coli was grown on Luria Broth (LB, Sigma-Aldrich, St. Louis, MO, USA) at 37°C under vigorous agitation, washed with phosphate buffer saline (PBS) 10 mM NaH2PO4, 2.68 mM KCl, 140 mM NaCl, pH 7.2 (Thermo Scientific, Waltham, MA, USA), resuspended at 3.6 × 10⁴ colony-forming unit (CFU)/mL, and homogenized using a glass homogenizer. Eight-month-old, canaries were immunized subcutaneously with Escherichia sp. in 50 µL (4, 1 × 10⁶ CFU per bird) of a water-in-oil emulsion containing 70% (w/w) Montanide™ ISA 71 VG adjuvant (Seppic, Paris, France), with a booster dose two weeks after the first dose. Control birds received a mock vaccine containing PBS and adjuvant. All reagents used for bacterial preparation were apyrogenic.

ExPEC strains JJ2050, JJ2528, and JJ2547 were used as parental isolates to develop phage resisters. These strains were kindly provided by James R. Johnson (6 ). In every case, these strains were grown overnight from a single colony in Luria broth (LB; Sigma-Aldrich) at 37°C and 250 rpm.

Transformation of competent E. coli DH5α was carried out following standard methods (22 -25 (link)). Briefly, approximately 50 ng of pUC19 were mixed with 50 µL of chemically competent DH5α on ice for 30 min. The mixture was subjected to heat shock at 42°C for 45 s, incubated on ice for 1 min, followed by the addition of 750 μL of liquid sterile Luria broth (LB) (Sigma, USA) for cell growth at 37°C for 1 h. Cells were harvested by centrifugation at 8,000 g for 5 min at room temperature, suspended with 100 μL of LB, and plated on LB agar containing 100 µg/mL of ampicillin, 1 mmol/L of IPTG and 20 µg/mL of X-Gal. Bacterial cells were grown on plates overnight at 37°C. On the next day, one colony of bacteria harboring pUC19 was selected based on the blue-white screening test and incubated in liquid LB under agitation at 230 rpm and 37°C for overnight growth. pUC19 plasmids were isolated using the Plasmid Plus Maxi kit (Qiagen, USA) according to the manufacturer instructions (26 ,27 ).

His_6x-tagged hRNF145 RING protein was produced in the bacterial RIPL strain (Novagen). Bacteria were grown in Luria Broth (LB) (Sigma) at 37°C to an OD₆₀₀ of 0.6 and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h. Bacterial pellets were collected and lysed in lysis buffer (50 mM Tris-HCl, pH 7.6, 0.5 M NaCl, 5 mM imidazole, and 1 mM DTT) supplemented with protease inhibitors and sonicated on ice to disrupt the cells. Debris was removed by centrifugation, and the cleared lysates were loaded onto HisTrap HP columns (GE Healthcare) coupled to an ÄKTAprime Plus protein purification system (GE Healthcare). Bound proteins were eluted with imidazole, buffer exchanged using Hi-Trap desalting columns (GE Healthcare), and collected in elution buffer (20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM DTT). Aliquots were immediately frozen in liquid N₂ and stored at −80°C.