Abi prism 377

Organoids were established from liver biopsies collected from patients and controls at the Hospital 12 de Octubre in Madrid (Spain) and also provided by Dr. Huch at Cambridge University (UK). The ZZ organoids were derived from ZZ AATD patients with hepatic failure who had liver transplant, whereas MZ organoids were obtained from an adult MZ AATD patient who underwent colicestomy. The control MM AAT organoids were derived from an individual with hepatocellular carcinoma undergoing surgical resection. Tissue sample was obtained from macroscopically defined non-neoplastic adjacent area. All biopsies were genotyped for SERPINA1. Sequencing of SERPINA1 gene coding exons was performed by using previously described primers [10 (link), 11 (link)] in an automatic sequencer (ABI PRISM 377 Applied BioSystems). Signed informed consent for the study was obtained from all the subjects and the research was approved by the ethics committee of Instituto de Salud Carlos III, Madrid, Spain.

PCR amplicon of ansA was purified by MinElute Gel purification Kit (Qiagen, Germany) and cloned into pTZ57R/T (MBI Fermentas), according to the manufacturer’s instructions. pTZ57R/T-ansA construct was transformed into competent E. coli JM109 (recA1, endA1, gyrA96, thi-1, hsdR17 (rK–mk+), e14–(mcrA−), supE44, relA1, Δ(lac-proAB)/F’ (traD36, proAB+, lac Iq, lacZ ΔM15) and plated on Luria Bertani (LB) agar containing ampicillin (100 μg mL⁻¹), isopropyl-β-D-thiogalactoside (IPTG, 50 μM) and X-gal (80 μg mL⁻¹) and incubated overnight at 37 °C. White colonies were selected for PCR amplification with vector primers M13f-M13r (MBI Fermentas) and the clones with correct insert as judged by size were sequenced on an ABI PRISM 377 genetic analyzer (Applied Biosystems Inc., USA).

The presence of integrons among APEC isolates was assayed using primers hep35 and hep36 that amplify conserved regions of integron-encoded integrase genes intI1, intI2, and intI3 using published PCR protocols [9 (link)]. Integron-positive isolates were characterized for the integron class using integron class-specific primers and PCR conditions previously described [10 (link)]. Further characterization of gene cassette content within integron classes was determined using primers 5′CS/3′CS and hep74/hep51 for amplification of gene cassettes within integron classes 1 and 2, respectively as published [11 (link), 12 (link)]. Samples with similar amplicon size were further subjected to restriction enzyme HinfI or RsaI [12 (link)] following manufacturer instructions (New England Biolabs, UK) and fragments obtained were separated on a 2.5% agarose gel. Isolates with similar polymorphism patterns were considered to harbor similar gene cassettes. Amplification products of samples with unique amplification products and one representative sample from samples with similar restriction patterns were purified from the agarose gel using QIA quick Gel Extraction Kit (Qiagen, USA) and sequenced using ABI Prism 377 automated sequencer (Applied Biosystems). All sequence results were compared with the available sequences in GenBank (http://www.ncbi.nlm.nih.gov/BLAST).

Standard molecular biology techniques were performed as previously described [72 ]. DNA fragments were purified with Gene-Turbo (BIO101 Systems). Plasmids and PCR products were purified with a High Pure Plasmid and PCR Product Purifications kits (Roche), respectively. Oligonucleotides were supplied by Sigma Co and they are detailed in Supplementary Materials Table S2. All cloned inserts and DNA fragments were confirmed by DNA sequencing with fluorescently labeled dideoxynucleotide terminators [73 (link)] and AmpliTaq FS DNA polymerase (Applied Biosystems) in an ABI Prism 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Transformations of E. coli were carried out by using the RbCl method or by electroporation (Gene Pulser, Bio-Rad, Hercules, CA, USA) [72 ]. Transformation of Azoarcus sp. CIB was done by biparental conjugation using the strain E. coli S17-1λpir as donor, following a protocol previously established [64 (link)] with slight modifications: donor cells were grown to A₆₀₀ of 5 and the receptor CIB strain was grown on MC medium supplemented with pyruvate 0.2%, and concentrated to reach an A₆₀₀ of 35. The transconjugants were selected on MC medium supplemented with 10 mM glutarate plus the corresponding antibiotic.

DNA sequencing reactions were performed on a plasmid template using the ABI Prism BigDye terminator cycle sequencing ready reaction kits (Applied Biosystems, California, USA) and an ABI PRISM 377 DNA sequencer (Applied Biosystems).