Noti restriction enzyme

Full-length COL7A1^c.425A>G was cloned by the Golden Gate cloning technique using NEBuilder^® Hifi DNA Assembly Cloning. The whole 9.2 kB cDNA was split into two fragments (F1: 4461 bp; F2: 4440 bp) and cloned into a pMXs-IRES-Blasticidin Retroviral vector (Cell Biolabs, San Diego, CA, USA) (F3: 5636 bp). Primers were designed using the NEBridge ^® Golden Gate Assembly Tool. Amplification of the fragments was performed using Phusion^TM High-Fidelity DNA Polymerase (ThermoFisher Scientific, Waltham, MA, USA) for F1 at an annealing temperature of 63.4 °C (forward primer: 5′-cctcgagggccggcgcgccgcATGACGCTGCGGCTTCTG-3′; reverse primer: 5′-cctttggggccAATAGCTCCAGGAGGTCCC-3′) and for F2 at an annealing temperature of 66.2 °C (forward primer: 5′-ctggagctattGGCCCCAAAGGTGACCGG-3′; reverse primer: 5′-gaggggcggaatttacgtagcTCAGTCCTGGGCAGTACCTG-3′). The vector was linearized with NotI restriction enzyme (New England Biolabs, Ipswich, MA, USA) and cloned according to the NEBuilder^® Hifi DNA Assembly Cloning (New England Biolabs, Ipswich, MA, USA) instructions.

Translation blocking antisense Morpholino (MOs; Gene Tools) against the ATG-containing sequence was designed (5′-AAATCCTCTGGGCATCTTCGCCAGC-3′) to target the translation start site according to the manufacturer's instruction and the other MO oligo (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was used as standard control. The MOs were diluted to 0.3 mM with RNase-free water and injected into the yolk of one to two-cell stage embryos and then raised in E3 medium at 28.5°C.
The cDNAs containing the open reading frame of the target genes were cloned into PCS2⁺ vector respectively and then were transcribed in vitro using the mMESSAGE mMACHIN Kit (Ambion, USA) after the recombinant plasmids linearized with NotI Restriction Enzyme (NEB, England), and then the capped mRNAs were purified by RNeasy Mini Kit (Qiagen, Germany). 2 nl target genes and mCherry mRNA mixture (1:1) were injected at 20 ng/μl into 1/2-cell stage embryos.

The de novo-synthesised chloramphenicol acetyltransferase (cat) gene from pBRT1Nc was amplified by polymerase chain reaction (PCR) using Q5 polymerase at 1 unit/µL (NEB, Hitchin, UK) and 5NotIdifcat and 3NotIdifcat primers (diluted 1:10 in sterile water from a stock solution of 100 pmol/µL). The DifCAT cassette that was generated was cut with a NotI restriction enzyme (NEB, Hitchin, UK) and ligated to generate Xer-cise plasmids pUCpW_difCAT and pUCpF_difCAT using Quick-Stick Ligase (Bioline Reagents Limited, London, UK) and Top10 E. coli competent cells (see Results) [41 (link)]. These were also amplified with primers rfbEdelF and rfbEdelR (designed with homologous 5′ sequences to rfbS and rfbX genes, respectively) and Q5 polymerase (NEB, Hitchin, UK) to create a rfbE chromosomal deletion cassette.

NotI restriction enzyme (New England Biolabs, United Kingdom) was used to analyze strains using a Pulse Net standardized PFGE protocol for V. cholerae (Cooper et al., 2006 (link)). The digested fragments were separated in 1% agarose (PFGE grade, Bio-Rad Laboratories, Inc.) prepared in a 0.5X TBE buffer in Clamped Homogenous Electric Fields Mapper (Bio-Rad Laboratories, Inc.). A standard running condition of the PulseNet protocol l¹ was used for the separation of DNA fragments. The gel was stained with ethidium bromide and photographed in a gel documentation system (Bio-Rad Laboratories, Inc., United States).
After visualization, the fingerprint profile in the PFGE banding pattern was analyzed by using the computer software package BioNumerics (7.1 versions) (Applied Maths, Belgium). The fingerprinting pattern, after background subtraction and gel normalization, was subjected to typing by band-based similarity Dice coefficient, which provides a quantitative assessment of strain similarity. Strains were clustered together using 1.5% optimization, 1.5% tolerance, and a threshold linkage value of >95% similarity matrix. Clustering was done based on unweighted-pair group methods using average linkages (UPGMA) as recommended by the software manufacturer, and results are graphically represented as a dendrogram.

The pmirGLO plasmid construction was based on the manufacturer manuals. The pmirGLO dual-luciferase miRNA target expression vector was purchased from Promega. The vector was digested with PmeI and XbaI restriction enzymes (New England Biolabs). The inserts for pmirGLO miR144 and pmiRGLO miR31 plasmids were purchased from Integrated DNA Technologies (Table S5) and were also digested with PmeI and XbaI restriction enzyme. The inserts were each ligated with digested pmirGLO plasmids using a quick ligasation kit (New England Biolabs) and transformed into JM109 competent cells (Promega). The plasmid was verified by digestion with the NotI restriction enzyme (New England Biolabs), according to manual.

The full-length coding gene sequence of asap1a or asap1b were amplified by using specific primers (Table 1) from the cDNA of 3 dpf zebrafish and subcloned into pCS2+ vectors using the Gibson Assembly enzyme-reagent mixture (NEB, Frankfurt am Main, Germany). The recombinant pCS2+-asap1a or pCS2+-asap1b plasmids were linearized using the Not I restriction enzyme (NEB). asap1a or asap1b mRNA were prepared by in vitro transcription using mMESSAGE mMACHINE™ SP6 Transcription Kit (Ambion, Austin, TX, USA) from pCS2+-asap1a or asap1b plasmids. The synthetic mRNA, at the concentration of 200 ng/μL, were co-injected into 1-cell stage embryos with asap1 morpholino mixture for rescue experiments according to the previous method (Rosen et al., 2009 (link)).