Bacterial genome extraction kit

A set of 108 salmonellae collected from multiple sources and stored at our laboratory was used in this study. Among these, 96 were the bovine isolates collected from dairy farms over various years (2007 to 2012) while 12 were the isolates collected from clinically sick humans by multiple hospitals around Zhejiang, Shenzhen and Shanghai (2011 to 2017). The bovine samples were collected during the regular epidemiological surveillance of animal disease, and from veterinary clinics at different geographical regions in the east coast, so there was no overlap between the humans and bovine isolates. No human patient personal data was available to the authors’ so informed consent or approval was not deemed necessary for the use of those isolates. The bovine isolates were identified to the genus level in the past by classical microbiological procedures whereas the human isolates were identified to the serovar level. All these isolates were re-confirmed to be true to the type using a specific multiplex PCR as recommended [41 (link)]. The genome for the downstream application was extracted using a commercial bacterial genome extraction kit (Tiangen Biotech, Beijing) and quantified using Nanodrop1000 (Thermo Fischer).

Genomic DNA of P. aeruginosa PAO1 was extracted by using a bacterial genome extraction kit (TIANGEN, China). The PaIDH1 (Gene ID: PA 2623) and PaIDH2 (Gene ID: PA 2624) genes were amplified with the primers in Tables S1 and S2. The PCR products with NdeI and XhoI (Thermo Scientific, Shanghai, China) digestion were cloned into expression vector pET-28b (+) to generate the recombinant plasmids pET-28b (+)-PaIDH1 and pET-28b (+)-PaIDH2. Mutations were introduced into PaIDH1 and PaIDH2 by overlap extension, PCR-based, site-directed mutagenesis.
The full-length gene of IDH K/P (AceK) from P. aeruginosa PAO1 was codon-optimized by selecting only the most preferential codons according to the E. coli bias and was cloned into the pET-28b (+) vector with the 6xHis label. GenScript Biotech Corp. (Nanjing, China) performed this procedure.
The correct sequences of each plasmid were verified by a sequencing service (General Biosystems, Hefei, China).

Aeromonas hydrophila AH3.5 was isolated from the intestinal tract of DR female Cx. pipiens pallens. Female mosquitoes were rinsed in 75% ethanol followed by sterile phosphate-buffered saline (PBS) to remove residual ethanol on the surface of the body. The midguts were then excised, ground and inoculated onto sterile Luria–Bertani (LB) agar plates using a three-zone marking method. Colonies that formed were purified by picking a single colony for expansion in liquid LB culture medium. This process was repeated 3 times to obtain stable and pure bacterial strains. A bacterial genome extraction kit from Tiangen Biochemical Technology Co., Ltd. (Beijing, China) was utilized, along with bacterial universal primers [Eub(27)F/Eub(1492)R] for PCR amplification. The amplified products were then forwarded to General Biotech Company for strain identification.
The isolated bacteria were mixed with 40% glycerol and stored at − 80 °C in a freezer in the insect room of the Department of Pathogen Biology, Nanjing Medical University for long-term preservation [9 ].

Genomic DNA of P. aeruginosa PAO1 was extracted by using a bacterial genome extraction kit (TIANGEN, China). The PaIDH1 (Gene ID: PA 2623) and PaIDH2 (Gene ID: PA 2624) genes were amplified with the primers in Tables S1 and S2. The PCR products with NdeI and XhoI (Thermo Scientific, Shanghai, China) digestion were cloned into expression vector pET-28b (+) to generate the recombinant plasmids pET-28b (+)-PaIDH1 and pET-28b (+)-PaIDH2. Mutations were introduced into PaIDH1 and PaIDH2 by overlap extension, PCR-based, site-directed mutagenesis.
The full-length gene of IDH K/P (AceK) from P. aeruginosa PAO1 was codon-optimized by selecting only the most preferential codons according to the E. coli bias and was cloned into the pET-28b (+) vector with the 6xHis label. GenScript Biotech Corp. (Nanjing, China) performed this procedure.
The correct sequences of each plasmid were verified by a sequencing service (General Biosystems, Hefei, China).

The LAB were grown overnight and the genomic DNA was extracted from 1.5 ml cultures using a bacterial genome extraction kit (Tiangen, China) following the manufacturer's protocols. The identification of isolates was analyzed based on the 16S rDNA gene amplification with primer pair: 16S rDNA-F (5′-AGA GTT TGA TCC ATG GCT CAG-3′)/16S rDNA-R (5′-AAG GAG GTG ATC CAG CC-3′). Then PCR was performed using a previously described method (24 (link)). The sequences for the amplified 16S rDNA were searched using Blast in the NCBI databases to compare with the registered sequences.

To obtain the genomic DNA of B. breve and B. longum, extraction was carried out according to the instructions of the bacterial genome extraction kit (Tiangen, China). Using the genomic DNA of B. breve and B. longum as templates, Primer Premier 5.0 was used to design primers based on the four esterase genes (b2, b3, b4, and bl) and the vector pET-28a. All of the primers (b2-F, b2-R, b3-F, b3-R, b4-F, b4-R, bl-F, bl-R, pET28a-F, and pET28a-R) are listed in Supplementary Table S2. By using the genomic DNA of B. breve and B. longum as templates, the four esterase genes were amplified by PCR and then purified along with the PCR-amplified vector DNA using the Gel Recovery Purification Kit (Tiangen, China). A Gibson Assembly Kit (NEB, United States) was used to insert the four esterase DNA fragments into the vector pET-28a to construct the recombinant vectors. Escherichia coli DH5α cells were transformed by the heat shock method and used for plasmid propagation to obtain the recombinant plasmids pET-28a-b2, pET-28a-b3, pET-28a-b4, and pET-28a-bl. All fragments were validated by Sanger 3730 sequencing.