E z n a plasmid mini kit 1

Genomic DNA from S. thermophilus 05-34 was prepared by lysozyme pretreatment and the genomic DNA Extraction Kit (Tiangen, Beijing, China). Mini-prep plasmid isolations from Lc. lactis were performed using the E.Z.N.A^TM Plasmid Mini Kit I (OMEGA Bio-tek Inc., Doraville, GA, United States). Standard PCR was carried out using Q5^TM High-Fidelity DNA polymerase following the manufacturer’s instructions (NEB, Beijing, China). DNA digestion with restriction endonucleases and DNA ligation were performed according to the manufacturer’s instructions (NEB). The electroporation of Lc. lactis NZ9000 was preformed according to previously described procedures (Holo and Nes, 1989 (link)). Primers used in this study were designed using PRIMER V5 software (PREMIER Biosoft International, Palo Alto, CA) and synthesized by Sangon Biotech (Beijing, China). DNA sequencing was performed by Sangon Biotech and the results were further analyzed with the DNAMAN software package (Lynnon Biosoftware, Vaudreuil, QC, Canada).

Each insert was assembled by incubating sfGFP_twist with an additional sequence acquired from Twist Bioscience, under BASIC linker ligation reaction conditions (21 (link)) in the presence of LMP-P and LMS-S linkers (Biolegio). Linker-ligated inserts were cloned into linker-ligated AmpR-pUC and transformed into E. coli DH5α. Resulting clones were incubated at 37°C overnight in LB media (ForMedium) supplemented with 50 μg/ml carbenicillin. Plasmid DNA was purified from cultures using E.Z.N.A.^® Plasmid Mini Kit I (Omega Bio-tek) and sequences verified by Source BioScience using SEVA_T0_rev and SEVA_T1_for primers. All plasmid sequences are available in the Supplementary Data.
Assembled expression cassettes were transformed into E. coli BL21(DE3) and picked colonies incubated in 200 μl LB medium supplemented with carbenicillin shaking overnight at 30°C. Overnight cultures were diluted 200 times into 100 μl LB supplemented with carbenicillin, 250 μM isopropyl β-D-1-thiogalactopyranoside and 0–2.5 mM theophylline. Cultures were grown shaking at 30°C to mid-log phase (6 h) and 2 μl off-sampled into 200 μl Phosphate Buffer Saline supplemented with 2 mg/ml kanamycin. Samples were analyzed for GFP fluorescence using a BD Fortessa flow cytometer and data analyzed using FlowJo_V10.

The recombinant DH5a E. coli carrying VTPIL-7 or VR1020 was cultured in routine LB broth containing kanamycin (100 mg/mL) in a shaker at 37 °C, 200 rpm for 12 h. They were then centrifuged, and the plasmids were isolated using the E.Z.N.A. ^® Plasmid Mini Kit I (OMEGA Bio-Tek, Guangzhou, China). The plasmids were resuspended in pure water and kept at –20 °C.
PEI/DNA complexes, Chitosan copolymer, CS-PEG-LAC and CS-PEG-PEI were prepared using the ionotropic gelation technique [22 (link),23 (link)]. In short, CS, CS-PEG-LAC and CS-PEG-PEI were respectively diluted with CH₃COOH/CH₃COONa (pH 5.5) fluid at a suitable ratio of triphosphate and heated to 65 °C for 10 min under slight magnetic mixing. A suitable quantity of chitosan solution was then mixed with the plasmid DNA solution, and the solutions were stirred well for 5 min. The nanoparticles were analyzed using Zetasizer 3000 HS/IHPL (Malvern Instruments Ltd., Malvern, UK).

PCR amplicons were subjected to agarose gel electrophoresis, excised from the gel, purified using an Agarose Gel DNA Purification Kit (TaKaRa, Japan), and cloned into the pMD18-T vector according to the manufacturer’s instructions (TaKaRa, Japan). The recombinant plasmid was transformed into DH5a competent E. coli cells. Plasmid DNA was purified using the E.Z.N.A.® Plasmid Mini Kit I (Omega Bio-Tek, USA) and quantified by spectrophotometric analysis. Recombinant plasmids of fragment A or fragment B were sequenced by Shanghai Sangon Bioengineering Ltd., China with primers included in Table-1. Detailed information, including the GenBank accession numbers of all sequences of the four novel strains, is available in Table-2. ClustalX software (version 1.83, Desmond G. Higgins, Paul Sharp, Trinity College Dublin, Ireland) was used to construct multiple alignments. Phylogenetic trees were constructed usingMEGAsoftware version 5.1 (https://mega.software.informer.com/5.1/), by the neighbor-joining analysis using the Maximum Composite Likelihood algorithm with 1000 bootstrap replicates. The datasets used for phylogenetic datasets included four novel FAdV-4 isolates from this study and 25 reference strains retrieved from GenBank database (Table-3).

The insertion sites of the colony-merger incompatible mutants were localized by plasmid rescuing and sequencing (Pan et al., 2010 (link)). Briefly, the genomic DNA of mutants was extracted using the cetyltrimethyl ammonium bromide method and digested with SacII. After purification by alcohol precipitation, fragments were self-circularized using T4 DNA ligase. The ligation product was transformed into the E. coli DH5α λpir strain. After cultivation on LB plates containing Km for ~14 h, five colonies were selected randomly for the extraction of plasmid DNA using the eZNA Plasmid Mini Kit I (Omega Bio-Tek) according to the manufacturer's instructions, and then sequenced using the primer 5′-GAA CTA TGT TGA ATA ATA AAA ACG-3′.

In silico studies were performed with BLAST (Altschul et al., 1990 (link)), NCBI (Sayers et al., 2022 (link)), and KEGG (Kanehisa and Goto, 2000 (link)) databases. PCR primers (Supplementary Table 2) were synthesized by Sigma-Aldrich Química SL (Madrid, Spain). Vector sequences were obtained from Addgene (Kamens, 2015 (link)). Vector or chromosomal DNA purifications were performed by miniprep with E.Z.N.A. Plasmid Mini Kit I (Omega Bio-tek, Norcross, Georgia) or DNeasyUltraClean Microbial Kit (Qiagen, Düsseldorf, Germany). Single-colony DNA was extracted by boiling and centrifugation. PCR products were purified using the ATP Gel/PCR DNA Fragment Extraction Kit (ATP Biotech Inc., Taipei, China). DNA was quantified with a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and sequenced by STAB VIDA (Caparica, Portugal).