Bacteria rna extraction kit

Total RNA was extracted from biofilm cells using Bacteria RNA Extraction Kit (Vazyme Biotech, Co., Ltd., China) according to the manufacturer’s instruction, and quantified by BioTek Synergy 2 (Waltham, MA, United States). Complementary DNA (cDNA) was synthesized through random hexamer primed reactions using a HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech, Co., Ltd., China). Afterward, RT-qPCR was performed in a final volume of 20 μL: 10 μL 2õ ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Co., Ltd., China), 1 μL forward and reverse primers, 2 μL template cDNA and 6 μL DNase/RNase-free ddH₂O. Primers used for RT-qPCR were shown in Supplementary Table S1. RT-qPCR reactions were carried out in a 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, United States) with the following profiles: 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 15 s, and annealing at 60°C for 60 s. Each PCR reaction was conducted in triplicate, and controls without template were included. Then the results calculated by normalizing target genes to respective reference gene based on the 2^–ΔΔCt method.

Total RNA extraction, removal of the genomic DNA and reverse transcription were performed using a Bacteria RNA Extraction Kit and HiScript III RT Supermix for qPCR (+gDNA wiper) kit (Vazyme) according to the manufacturer’s instructions. SYBR Premix ExTaqTM (Takara, Dalian, China) was used for qRT–PCR, and the cycle thresholds were determined using a Roche LightCycler® 480 II sequence detection system (Roche, Shanghai, China). rnpB (RNase P subunit B) was used as the internal control. The primers for alr3614 (cvkR) and rnpB are listed in Supplementary Table 3. Three independent experiments were performed, which showed consistent results.

The absolute expression of hns and tet(X4) was detected by RT-qPCR. Firstly, hns and tet(X4) genes were, respectively, cloned into pBAD and pCE2 to generate pBAD-hns and pCE2-tet(X4). Plasmid pBAD-hns and pCE2-tet(X4) were used as template DNA with primers hns-RNA-F, hns-RNA-R, tet(X4)-RNA-F, and tet(X4)-RNA-R, respectively, to set up the corresponding standard curve. The total RNA of bacteria was extracted by Bacteria RNA Extraction Kit (Vazyme Biotech Co., Ltd) and then reverse transcribed into cDNA by 1 μg of RNA using the HiScriptR^R III RT SuperMix for qPCR (+gDNA wiper) Kits (Vazyme Biotech Co., Ltd), and the cDNA of 1 μl was used as template for RT-qPCR with primers hns-RNA-F, hns-RNA-R, tet(X4)-RNA-F, and tet(X4)-RNA-R using the ChamQTM Universal SYBRR^R Color qPCR Master Mix Kits (Vazyme Biotech Co., Ltd). According to the standard curve, the absolute expression of hns and tet(X4) genes was calculated. The specific sequence of the primers is shown in Supplementary Table 3.

For induced strains with at least a four-fold increase in the MIC values of CAZ-AVI, the transcription levels of bla_KPC of all passages were measured_. For each strain, overnight culture was inoculated 1:1,000 into 4 ml of fresh LB broth with CAZ-AVI at 1/2 of the MIC for the original strain and cultured at 37°C with 220 rpm shaking until the growth reached the logarithmic growth phase. Total RNA was isolated using a Bacteria RNA Extraction Kit (Vazyme, Nanjing, China) and cDNA was produced using a HiScript III-RT SuperMix for qPCR kit (Vazyme, Nanjing, China) according to the manufacturer's instructions. RT–PCR was performed using a ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a LightCycle® 96 (Roche) using bla_KPC primers (Table S1). The relative transcript levels were calculated using the 2^-ΔΔCT method [41 (link)] with rpoB as the reference (Table S1). The average transcript levels were calculated from at least three independent RNA samples isolated from three separate microbial broth cultures for each strain.
The previous study showed that LamB plays an important role in OmpK35/OmpK36-defective and OmpK36-defective strains, thus, the transcription levels of proins (ompK35, ompK36, and ompK37) were also detected to explore the role of LamB [42 (link)] using the method described above.

The CRISPR interference system was used to generate a mutant Shigella sp. PIB with knockdown of KS. The pdCas9 vector (Addgene, 44249) was used as a vector backbone. A fragment containing a J23119 promotor (5′-TTGACAGCTAGCTCAGTCCTAGGTATAATACTAGT-3′) and a guide DNA (gDNA) of KS (5′-TTACATTAAATATTACCGACTGG-3′) plus an sgRNA coding template was inserted upstream of the dCas9 coding sequence through a BglII site. The resultant vector (J23119 + KS gDNA + sgRNA coding DNA + J23119 + dCas9) persistently expressed the KS gDNA and dCas9, decreasing KS gene expression in bacteria. The KS gene knockdown bacterium (PIB-KD) was generated by electrotransformation (1800 W, 25 μF, 200 Ω, Bio-Rad) with the vector and selection with chloramphenicol (10 μg/mL). To verify the knockdown efficiency, the total RNA of the mutant bacterium was isolated with a Bacteria RNA Extraction Kit (R403-01, Vazyme Co.) and reverse transcribed to cDNA (Hiscript Q RT SuperMix for qPCR [+gDNA wiper] kit, R123-01, Vazyme Co.). The expression of the KS gene was quantified with a Taq Pro Universal SYBR qPCR Master Mix kit (Q712-02, Vazyme Co.). The expression of 16S rDNA was used as internal control.

Quantitative reverse-transcription PCR was used to examined the transcriptional levels of different genes in the WT and mutant strains. Overnight cultures of WT, ΔvmeL, and CΔvmeL V. parahaemolyticus were adjusted to an OD₆₀₀ of 0.2, and 2.5 μL aliquots were spread separately onto swarming plates. Cells were collected from swarming plates after 15 h of cultivation, and were suspended in 1 mL of TRIzol (Yu et al., 2019 (link)). Planktonic bacteria were cultivated in liquid MLB medium to the logarithmic phase with shaking at 37°C, and the swarming cells were collected. Then, total RNA was isolated from each sample following manufacturer protocol (Bacteria RNA Extraction Kit, Vazyme, Nanjing, China). RNA was reverse transcribed to cDNA. cDNA was utilized as a template for quantitative qRT-PCR. For each gene, reactions were performed for three RNA samples and each reaction was performed in triplicate. The transcriptional level was quantified using the 2^–ΔΔCt method using the housekeeping gene gapA (Genbank: BAC61233.1) for normalization (Livak and Schmittgen, 2001 (link)). The primers for qRT-PCR are provided in Supplementary Table 1.