All purified DNA samples and peripheral venous blood samples were initially diagnosed using a thalassemia diagnostic kit (Hybribio, China) by reverse dot blot method and further verified by DNA sequencing (Invitrogen, China). For the verification of allele genes, the ALDH2 gene from buccal swabs was detected by DNA sequencing (Invitrogen, China). Bacteria in urine samples were cultured on the blood agar medium plate (Pangtong, China) and then diagnosed by the matrix-assisted laser desorption ionization–time of flight mass spectrometry (bioMérieux, France).
Dna sequencing
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
DNA sequencing is a laboratory technique used to determine the precise order of the four chemical building blocks (adenine, guanine, cytosine, and thymine) that make up a DNA molecule. This process provides valuable information about the genetic makeup of an organism.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 plasmid, by Thermo Fisher Scientific (2 mentions)
Mir 497 5p mimic, by RiboBio (2 mentions)
Mir 497 5p inhibitor, by RiboBio (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Matrix assisted laser desorption ionization time of flight mass spectrometry, by bioMérieux (1 mentions)
Rotor gene q6000, by Qiagen (1 mentions)
Rotor gene 6000, by Qiagen (1 mentions)
Beacon designer, by Premier Biosoft (1 mentions)
Syber green master mix, by Takara Bio (1 mentions)
Axyprep blood genomic dna miniprep kit, by Corning (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Pmir report vector, by Thermo Fisher Scientific (1 mentions)
Pjet1.2 vector, by Thermo Fisher Scientific (1 mentions)
Luria bertani medium, by Thermo Fisher Scientific (1 mentions)
Plasmid maxi kit, by Qiagen (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Pentobarbital sodium, by Merck Group (1 mentions)
Sybr qpcr mix, by Thermo Fisher Scientific (1 mentions)
32 protocols using dna sequencing
1
Comprehensive Genetic Diagnostics Protocol
2
Construction of Isogenic K. pneumoniae Mutants
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K. pneumoniae NTUH-K2044 (a strain that resulted in pyogenic liver abscess in a 66 year old patient) was kindly provided by Dr. Jin Town Wang of the National Taiwan University Hospital, Taipei, Taiwan [10] (link). E. coli SM10 lambda pir and pUT-Km was used to create isogenic mutants. Genomic DNA, plasmid, restriction digestion, DNA elution (Qiagen), ligation, transformation, conjugation, DNA sequencing (Applied Biosystems) were performed as previously described [22] (link), [23] (link), [24] . Primers used in the present study were custom-synthesized (Eurofins MWG operons, Germany).
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K. pneumoniae NTUH-K2044 (a strain that resulted in pyogenic liver abscess in a 66 year old patient) was kindly provided by Dr. Jin Town Wang of the National Taiwan University Hospital, Taipei, Taiwan [10] (link). E. coli SM10 lambda pir and pUT-Km was used to create isogenic mutants. Genomic DNA, plasmid, restriction digestion, DNA elution (Qiagen), ligation, transformation, conjugation, DNA sequencing (Applied Biosystems) were performed as previously described [22] (link), [23] (link), [24] . Primers used in the present study were custom-synthesized (Eurofins MWG operons, Germany).
3
Quantitative Real-Time PCR Analysis of Bcl-2 and Bax
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The primers of quantitative real time PCR Syber Green method were designed for complete cDNA of Bcl-2, Bax, and β-actin based on sequence data available on NCBI databases utilizing Beacon Designer (PREMIER Biosoft International, Palo Alto, CA; version 7). Primers’ specificity were checked by BLAST analysis (NCBI). A quantitative real-time PCR was done by Syber Green Master Mix (Takara Biotechnology, Otsu and Shiga, Japan). The primers’ specificity and reliability was approved via performing endpoint PCR and amplicon sequencing through DNA sequencing, Applied Biosystems (SEQLAB, Germany). The designed primers are summarized in Table 1 . β-actin was utilized as housekeeping gene for the normalization of data. Relative two standard curves real time PCR Syber Green method analyzed gene expressions in a Rotor Gene Q 6000 (Qiagen Hilden, Germany). The data were analyzed using the 6-point 10-fold serial dilution standard curves for genes of interest and reference gene. Standard curves were plotted for target and reference genes, and then Rotor Gene 6000 software (Qiagen, Germany) was used to analyze the data. The relative quantity of Bcl-2 and Bax gene expressions were normalized to the β-actin mRNA relative quantity and expressed as gene expression index.
4
Construction of pZBD-chimeras using Ec-GluRS
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A previously reported [15 (link)] Ec-GluRS encoding plasmid was used for the construction of pZBD-chimeras. Four sets of oligonucleotides were designed and were purchased from Integrated DNA Technologies. Using the above Ec-GluRS plasmid as a template, two separate sets of PCR reactions were performed with appropriate oligonucleotide combinations. The overall construction scheme along with the adequate oligonucleotide sequences is shown in Supplementary Figure S1. The resultant PCR products were cloned separately in the pETSUMO2 vector, as described previously [15 (link)]. All constructs were confirmed by DNA-sequencing (Applied Biosystems) and purified as described previously [15 (link)].
5
Genotyping SNPs in FoxO1, A2M, and TGF-β1
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Single nucleotide polymorphisms (SNPs) in the FoxO1 (rs2297626, rs7319021, rs28553411, rs17592468, and rs17592371), A2M (rs10492115, rs226415, rs10842849, rs11048839, rs10842847, and rs669), and TGF-β1 (rs2317130, rs1800469, rs12983775, rs12462166, and rs2241715) genes were selected for genotyping, the candidate SNPs comprised most of the allelic variants with r2 > 0.8 in the Asian population. Genomic DNA was extracted from venous blood samples using the AxyPrep Blood Genomic DNA Miniprep kit (Axygen, Union City, CA, USA). Polymerase chain reaction (PCR) was performed in a reaction volume of 2 μl DNA, 7.5 μl 2× PCR mix, 2 μl primer mix, 0.2 μl ExoI enzyme (Fermentas, Burlington, ON, Canada), 0.7 μl ExoI buffer (Fermentas), and 0.8 μl FastAP enzyme (Fermentas). Amplification conditions were as follows: at 95°C for 3 min; 35 cycles of 94°C for 15 s, 55°C for 15 s, and 72°C for 30 s; and 72°C for 3 min. PCR products were purified by incubation with ExoI and FastAP at 37°C for 15 min and at 80°C for 15 min. Extension conditions were as follows: 96°C for 1 min, and 30 cycles of 96°C for 10 s, 52°C for 5 s, and 60°C for 30 s. Extension products after denaturation at 95°C for 3 min were analyzed by DNA sequencing (Applied Biosystems, Foster City, CA, USA).
6
miRNA Reporter Vector Construction
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To generate reporter vectors bearing binding sites for the three miRNA examined in detail, miR-98, miR-212 and miR-137, oligonucleotides encoding target gene miRNA recognition elements (MREs) were annealed to form SpeI and HindIII restricted overhangs of a ligatable cassette compatible with SpeI and HindIII digested pMIR-REPORT vector (Ambion, Austin, TX) as described previously
[69 (link), 70 (link)]. Recombinant reporter constructs were amplified in chemicompetent DH5alpha Escherichia coli bacteria cells (Bioline, Sydney) and purified using endotoxin free minipreps (Promega, Madison, WI) before being verified by DNA sequencing (AGRF, St. Lucia, QLD, Australia) and quantitated using a NanoDrop 2000 (Thermo Scientific, Delaware, ME, USA).
[69 (link), 70 (link)]. Recombinant reporter constructs were amplified in chemicompetent DH5alpha Escherichia coli bacteria cells (Bioline, Sydney) and purified using endotoxin free minipreps (Promega, Madison, WI) before being verified by DNA sequencing (AGRF, St. Lucia, QLD, Australia) and quantitated using a NanoDrop 2000 (Thermo Scientific, Delaware, ME, USA).
7
Cas12a-Mediated Site-Specific PCR Amplification
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The primers used for PCR are listed in Table 1 . Two thymine bases (TT) were inserted in the appropriate site of the forward primer, to introduce the Cas12a-recognized PAM (Sequences: TTTC). PCR was performed in a 20 μl reaction solution containing 4 μl of 5 × PrimeSTAR Buffer, 1.6 μl of dNTP mixture (10 mM), 0.4 μl each of forward and reverse primer (10 μM), 0.2 μl of DNA polymerase, 0.8 μl of DNA template, and 13 μl of ddH2O. The amplification protocol was as follows: 98°C for 5 min, 30 cycles of 98°C for 10 s, 60°C for 5 s, and 72°C for 15 s; and a final step of 72°C for 5 min. The amplified products were electrophoresed on a 1.5% agarose gel and cloned into the pJET1.2 vector (Thermo Fisher Scientific, USA), followed by DNA sequencing (Tsingke Biotechnology Co., Ltd.).
8
Expression and Purification of Der f 23 Allergen
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Der f 23 open reading frame (ORF) cDNA was amplified by polymerase chain reaction (PCR) using the Primer STAR HS DNA polymerase with the D. farinae cDNA library. The primers used were: Forward, 5′-ATGAAATTCAACATAACTATCGC-3′; and reverse, 5′-TTATGTACATGTTAATTCTTTTTCA-3′. The PCR thermocycling conditions were 94°C for 30 sec, 55°C for 30 sec and 72°C for 50 sec, for 30 cycles. The PCR product, confirmed by DNA sequencing (GenScript Biotech Corporation), was subcloned into a pET-His vector, and then transformed into Escherichia coli (E. coli) BL21 (DE3) plysS. E. coli were grown overnight in Luria-Bertani medium (Thermo Fisher Scientific, Inc.) containing 100 mg/l ampicillin at 37°C, and were induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Following cultivation for additional 3 h at 37°C, E. coli cells were harvested by centrifugation at 9,600 × g for 5 min at 4°C. Following mixing with the protein extraction buffer (20 mM PB, 150 mM NaCl and 1 mg/ml lysozyme), the harvested cells were sonicated for 3 sec with 5-sec intervals in an ice bath for a total of 20 min, and then centrifuged again at 9,600 g for 20 min at 4°C. The recombinant protein in the soluble portion was purified with a Ni-NTA column (cat. no. 17040303; GE Healthcare) and gel filtration (HiLoad Superdex 16/600; cat. no. 28-9893-33; GE Healthcare).
9
Engineered Bacillus Strains for Protein Expression
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Gene cloning and site directed mutagenesis was done using standard molecular biology procedures as described before14 (link), 22 (link) and B. anthracis Sterne strain genomic DNA. The clones were confirmed with restriction digestion and DNA sequencing (Invitrogen). Site-directed mutagenesis was carried out using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) using GroEL clones as templates. The details of primers and plasmids are provided in Table S1 .
Genes encoding for PrkC (1-657 aa), wild-type GroEL (1-544 aa), and GroEL mutants (GroEL-T21A, GroEL-T132A, and GroEL-T329A) were cloned into the E. coli, B. anthracis shuttle vector, pYS5 (modified to express Spectinomycin resistance gene63 (link)), and electroporated in B. anthracis Sterne strain (Bas-wt or BasΔprkC strain) (BTX Electro Cell Manipulator 600).
Genes encoding for PrkC (1-657 aa), wild-type GroEL (1-544 aa), and GroEL mutants (GroEL-T21A, GroEL-T132A, and GroEL-T329A) were cloned into the E. coli, B. anthracis shuttle vector, pYS5 (modified to express Spectinomycin resistance gene63 (link)), and electroporated in B. anthracis Sterne strain (Bas-wt or BasΔprkC strain) (BTX Electro Cell Manipulator 600).
10
Site-directed Mutagenesis of BdsA Protein
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Site-directed mutagenesis of BdsA was conducted by two-step PCR using the wild-type pET-15b/bdsA plasmid as template. The mutant clones were confirmed by DNA sequencing (Invitrogen). The expression and purification processes were the same as that for the wild-type BdsA protein.
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