Bacterial dna kit

Total DNA was extracted from fecal samples using the bacterial DNA Kit (D3350-02, Omega Biotek, Norcross, GA, USA). After extraction, DNA integrity and quality was checked by 1% agarose gel electrophoresis and a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, USA). PCR reaction were performed in 20 μl volumes, containing 4 μl Fast pfu buffer, 2 μl 2.5 mM dNTPs, 0.8 μl each primer (5 μM), 0.4 μl Fast pfu polymerase, 0.2 μl bovine serum albumin (BSA) and 10 ng template DNA. The V3–V4 regions of bacterial 16S rDNA was amplified with using the primers, 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Yang et al., 2019 (link)). PCR parameters were 3 min denaturation at 95°C, 29 cycles of 30 s at 95°C, 30 s annealing at 53°C, 45 s elongation at 72°C and a final extension at 72°C for 10 min.

DNA was extracted from isolates using a Bacterial DNA Kit (Omega Bio-Tek) following the protocol of the manufacturer. PCR of 16S rRNA genes and sequencing were conducted by Majorbio Company (Shanghai, China). In brief, the almost complete bacterial 16S rRNA gene was amplified with Ex Taq^® (Takara Bio Inc.), using primers 27F (5’-AGAGTTYGATCCTGGCTCAG-3’) and 1492R (5’- GGTTACCTTGTTACGACTT-3’). Conditions comprised an initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 1.5 min, and a final extension step at 72°C for 10 min on the last cycle. The amplicons were purified and sequenced on a 3730XL DNA analyzer system (Applied Biosystems). Retrieved DNA sequences were manually edited using Seqman Pro (DNASTAR Lasergene package). The similarities of 16S rRNA genes between isolated endophytes and their phylogenetic neighbors were determined through the EzBioCloud Database (access time: January 2023) (Yoon et al., 2017 (link)), supplemented with the BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of the NCBI to search for the closest matching sequences.

Cells were harvested when the OD₆₀₀ was 1.0, and then a Bacterial DNA Kit (Omega Biotek, USA) was used to isolate genomic DNA. The quality of genomic DNA was checked via 0.7% agarose gel electrophoresis run for 45 min at 120.0 V/cm.
Genomic DNA of the 10 genome-shuffled mutants and parental strain F34 was sequenced by an Illumina HiSeq instrument (Illumina, San Diego, CA, USA), and the reference genome of strain ZM4 (GenBank No. NC_006526.2) was mapped. Annotation for potential SNVs, indels, and SVs was performed by ANNOVAR (V21 Feb 2013). The sequencing was completed by GenWize, Inc. (Suzhou, China).
Fermentation supernatant was centrifuged at 13,500 rpm for 5 min, and the precipitate was discarded; the supernatant was then passed through a 0.22 μm membrane and used to determine the concentrations of glucose and ethanol in the fermentation. High-performance liquid chromatography (HPLC, Agilent 1200) was applied to assess glucose and ethanol concentrations with 5 mM H₂SO₄ at a flow rate of 0.6 mL/min and a column temperature of 35 °C. The injection volume was set at 20.0 μL. The cell density was determined by a spectrophotometer detector (Jingke UV765, Shanghai) at wavelength 600 nm. Differences between the fermentation profiles of each genome-shuffled strains and the control strain were tested by one-way ANOVA.

After being incubated, twenty individual colonies were randomly selected from 2216E agar plates of each ark clam and sub-cultured for identification. Bacterial DNA was extracted using the Bacterial DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. The preliminary identification of isolated bacteria was performed using the primers 27F and 1492R to amplify 16S rDNA (Table 1) [44 (link)]. In addition, the housekeeping genes atpA (ATP synthase alpha subunit gene), mreB (rod shaping protein B subunit gene), pyrH (uridylate kinase gene), recA (recombinase A gene), and rpoA (RNA polymerase alpha subunit gene) were amplified and sequenced, as previously described, to further confirm the species of isolated vibrios [45 (link)]. The concatenated sequences of atpA-mreB-pyrH-recA-rpoA from the isolates and other reported Vibrio strains were aligned using the CLUSTAL_W tool and the phylogenetic tree was evaluated using bootstrap analysis based on the neighbor-joining (NJ) method with 1000 replicates, which was embedded in MEGA version 7.0.

Gut samples were taken from healthy and diseased hybrid sturgeon, and bacterial genomic DNA was extracted using a bacterial DNA kit (Omega Biotek, Winooski, VT, USA). The extracted DNA was used as a template for the amplification of the V3–V4 high variable region of the 16S rRNA gene using the GeneAmp 9700 system (Applied Biosystems, Foster City, CA, USA). The primers were 341F 5′-ACTCCTACGGGAGGCAGCAG-3′and 806R 5′-GGACTACHVGGGTWTCTAAT-3′ (Xu et al., 2022 (link)).Thermal cycling comprised an initial denaturation at 95°C for 2 min, the amplification conditions were 30 cycles of 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; with a final extension at 72°C for 10 s. After checking the DNA quality using 1% agarose gel electrophoresis, the samples were sequenced on the Illumina MiSeq PE300 high-throughput sequencing platform (Illumina Inc., San Diego, CA, USA). The composition of the gut microbiota was analyzed at the phylum and genus levels in the healthy and diseased groups using the R software. Alpha-diversity indicators and beta-diversity indicators of the gut microbiota were calculated at the website (http://www.genescloud.cn/analysisProcess). Principal coordinates analysis (PCoA) was used to analyze the operation taxonomic units (OTUs) for each sample.