Zr fungal bacterial dna miniprep

Pure cultures on SDA plates were harvested by flooding each plate with 3 mL of phosphate buffered saline (PBS, pH 7.4) followed by gentle scraping of the agar surface with an L-shaped glass Pasteur pipette. The suspension was then collected into a 15 mL centrifuge tube with PBS washed glass beads and vortex-mixed for 5 min. For DNA extraction, 200 µL of suspension was processed with the ZR Fungal/Bacterial DNA MiniPrep™ (Zymo Research, USA) according to the protocol provided by the manufacturer.

Bacterial genomic DNA was obtained from pure colonies of the PEPV40 strain grown on YMA plates and collected after 24 h at 28 °C using the ZR Fungal/Bacterial DNA MiniPrep (Zymo Research). The draft genome sequence of the bacterial isolates was obtained by shotgun sequencing on an Illumina MiSeq platform via a paired-end run (2 × 251 bp). The sequence data was assembled by Velvet 1.2.10 and gene annotation was performed using RAST 2.0 (Rapid Annotation using Subsystem Technology) [18 (link)].
The draft genome sequence of the strain Rhizobium laguerreae PEPV40 was deposited in GenBank under the accession number JABWPR000000000. The draft genome sequence of the strain Bacillus halotolerans SCCPVE07 was obtained previously in Jiménez-Gómez et al. [11 (link)].

The genomic DNA for genome sequencing was obtained from bacterial cells of strains IA19^T, A2-NA12, and A2-NA13 grown on NA plates and collected after 24 h at 28°C, using the ZR Fungal/Bacterial DNA MiniPrep (Zymo Research).
The draft genome sequences of the selected isolates were obtained by shotgun sequencing on an Illumina MiSeq platform via a paired-end run (2 × 251 bp). The sequence data were assembled using Velvet 1.2.10 (Zerbino and Birney, 2008 (link)) and a draft genome was obtained. Gene calling and annotation was performed using RAST 2.0 (Rapid Annotation using Subsystem Technology) (Aziz et al., 2008 (link)). The SEED-viewer framework (Overbeek et al., 2014 (link)) was used for a first mining of genes related to antimicrobial production genes. Moreover, a more specific and detailed analysis of the presence of gene clusters related to antimicrobial substances productions and other secondary metabolites was performed using antiSMASH 3.0 (Weber et al., 2015 (link)).

The amplified DNA (60 μL of ddMDA or 20 μL of tube MDA) was cleaned using a DNA Clean & Concentrator™-5 (Zymo Research, Irvine, CA) and the control genomic DNA was prepared from 10ml of overnight culture of E.coli K-12 MG1655 using ZR Fungal/Bacterial DNA MiniPrep™ (Zymo Research) by following the manufactures’ protocols. After quantification using Qubit® dsDNA HS Assay Kit (Life Technologies, San Diego, CA), one nanogram each of purified DNA from the MDA samples and one nanogram of genomic DNA were prepared for a sequencing library using Nextera XT kit (Illumina, San Diego, CA) and pooled together as the manufacture’s protocol. Sixteen picomolar of the pooled library was loaded to MiSeq (Illumina) for a 75-cycle paired-end reads using the v3 chemistry.

Plasmids for the expression of the endolysins encoded by the phages bIL285 and bIL286 were constructed by generating PCR fragments of the genes pi252 and pi305, respectively (Phusion Hot Start II, Thermo Fisher Scientific, Wilmington, Delaware USA) by using L. lactis subsp. lactis IL1403 genomic DNA as a template (ZR Fungal/Bacterial DNA MiniPrep, Zymo Research, Irvine, CA, USA) with primers bil285F/bil286R and bil286F/bil286R. PCR fragment of pi252 was digested with BsaI to generate cloning overhangs and EcoRV to digest co-amplified pi149 and pi305 PCR fragments. PCR fragment pi305 was digested with BsaI, ApaI and AflII restriction enzymes (New England Biolabs, Ipswich, MA, USA) to generate cloning overhangs and digest co-amplified pi149 and pi252 PCR fragments, respectively. Both digested PCR fragments were inserted into NcoI/HindIII-linearized vector pNZ8048e. PCR products were purified by using the High Pure PCR Purification Kit (Analytic Jena, Jena, Germany). Ligations were performed by using T4 DNA Ligase (New England Biolabs), and the resulting plasmids were transferred to electrocompetent L. lactis PA1001 as described before (Leenhouts and Venema 1993 ). All selected plasmids were checked by sequencing (Eurofins MWG Operon, Ebersberg, Germany).

Dietzia sp. D5 isolated in our laboratory is deposited in Culture Collection, University of Göteborg (CCUG 64924), Sweden and its genomic DNA was purified using ZR Fungal/Bacterial DNA MiniPrep (Zymo Research, Irvine, USA). E. coli NovaBlue, BL21(DE3), Rosetta2(DE3) and the plasmids pET-22b(+) were purchased from Novagen (Darmstad, Germany). E. coli BL21-CodonPlus(DE3)-RP and ArcticExpress(DE3)-RP cells were purchased from Agilent Technologies (Santa Clara, USA).