Escherichia coli strain TOP 10 (Invitrogen, Carlsbad, CA, USA) and the destination vector pPZP200/Ubi-HA-ccdB-Nos-35S-hpt-tml (Chiang et al., 2017) were respectively used for gene construction and transformation. Briefly, HPV52L1 was constructed by the Invitrogen pENTR/D-TOPO Expression Vector System ® for plant transformation. HPV-52L1 was amplified with the above-described paired primers (HPV52L1-5F and -3R) from the RT-PCR using high-fidelity DNA polymerase (New England Biolabs, Beverly, MA, USA). HPV52L1 fragments were amplified to 1.59 kb, in which water was used as a negative control. The PCR product was purified using the SNAP Gel Purification Kit (Invitrogen), ligated to the pENTR TOPO vector, and transformed into TOP10. After pENTR cloning, the constructed plasmid, named pENTR-OsHPV52L1 (Supplemental Figure S1A), was cloned into the destination vector, pPZP200/Ubi-HA-ccdB-Nos-35S-hpt-tml. After Spectinomycin screening of the colony, plasmid insertion was confirmed by PCR, and DNA sequences of the pPZP200/Ubi-HA-HPV52L1-Nos (pPZP200-OsHPV52L1) clones were confirmed (Supplemental Figure S1B).
Top10
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
TOP10 is a competent laboratory strain of Escherichia coli, commonly used in molecular biology applications. It is designed to facilitate the cloning and propagation of recombinant DNA.
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Bl21 star de3, by Thermo Fisher Scientific (4 mentions)
Bl21 de3, by Agilent Technologies (3 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (3 mentions)
E coli dh5α, by Thermo Fisher Scientific (3 mentions)
Yeast extract, by BD (2 mentions)
Todd hewitt broth, by BD (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Erythromycin, by Merck Group (2 mentions)
Neb5α, by New England Biolabs (2 mentions)
Qiaprep miniprep kit, by Qiagen (2 mentions)
Dh10b, by Thermo Fisher Scientific (2 mentions)
Rosetta 2, by Merck Group (2 mentions)
Kanamycin, by GoldBio (2 mentions)
Uw2070, by Bandelin (2 mentions)
Econo pac 10dg column, by Bio-Rad (2 mentions)
Bl21 de3, by Thermo Fisher Scientific (2 mentions)
Ampicillin, by Thermo Fisher Scientific (2 mentions)
Spectinomycin, by Thermo Fisher Scientific (2 mentions)
Pdest14, by Thermo Fisher Scientific (2 mentions)
Gwizblank, by Aldevron (2 mentions)
127 protocols using top10
1
Construction of HPV52L1 Expression Vector
2
Escherichia coli and Ustilago maydis Strains
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The Escherichia coli strain Top10 (Life technologies) and BL21(DE3)pLysS (Promega) were used for cloning purposes and for expression of recombinant Ros1 protein respectively. U. maydis strains used in this study are listed in S5 Table , they are derivates of haploid strains FB1 and FB2 [77 (link)] or AB33 [39 (link)]. Cells were grown in liquid YEPSL (0.4% yeast extract, 0.4% peptone, 2% sucrose) at 28°C on a rotary shaker at 220 rpm. For virulence assays, compatible haploid strains were grown separately in YEPSL to an OD600 of 1.0, transferred to the same volume of sterile water and mixed in equal amounts prior to injection into maize seedlings.
For premature ros1 expression studies, AB33 and strains derived from AB33 were grown in complete medium (CM) [78 ] supplemented with glucose (2%) to an OD600 of 0.5. Cells were collected by centrifugation, washed with H2O and resuspended in nitrate minimal medium (NM) [78 ] containing arabinose (2%) as sole carbon source. Cells were subsequently grown for 12h for microscopic observation. To induce ros1 after the switch to filamentous growth, AB33 or AB33 derived strains were incubated for 6 h in NM + glucose and then shifted to NM + arabinose for 6 h. All chemicals used for media preparation were of analytical grade and were obtained from Sigma-Aldrich.
For premature ros1 expression studies, AB33 and strains derived from AB33 were grown in complete medium (CM) [78 ] supplemented with glucose (2%) to an OD600 of 0.5. Cells were collected by centrifugation, washed with H2O and resuspended in nitrate minimal medium (NM) [78 ] containing arabinose (2%) as sole carbon source. Cells were subsequently grown for 12h for microscopic observation. To induce ros1 after the switch to filamentous growth, AB33 or AB33 derived strains were incubated for 6 h in NM + glucose and then shifted to NM + arabinose for 6 h. All chemicals used for media preparation were of analytical grade and were obtained from Sigma-Aldrich.
3
Fission Yeast Strain Selection Protocol
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All the S. pombe strains used in this study are shown in Table 1 . Selection for strains containing telomere cassettes was performed in Edinburgh minimal medium with sodium glutamate substituted for ammonium chloride (EMMG) without uracil and with appropriate amino acid supplements and 100 μg/ml HygroGold (InvivoGen) (81 (link)). Nonselective growth of strains bearing the telomere cassettes was done in EMMG with adenine, histidine, uracil, leucine, lysine, and arginine (EMMG plus AHULKR) and without hygromycin. Preparation of 10 mM ahTET stock and plates was performed as described previously (34 (link)). 5-Fluoroorotic acid (5-FOA) plates were yeast nitrogen base plates with 1 mg/ml 5-FOA (Toronto Research Chemicals, Inc.) (http://www-bcf.usc.edu/~forsburg/drugs.html ) and with the appropriate supplements. All recombinant DNA procedures were carried out in NEB 5-alpha (New England BioLabs) and TOP10 (Life Technologies) competent cells.
4
Streptococcus mutans Growth and Escherichia coli Propagation
5
CRISPR Vector Construction for SPATA31 Targeting
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10 μg of pX260 or pX330 were digested with BbsI (NEB) for 3 h at 37 ° C. Digested pX260 or pX330 vectors were purified by QIAEXII Extraction Kit (Qiagen) according to manufacturers recommendation. Targeting guide RNA sequence specific to exon1 of SPATA31 genes was designed by using the CRISPR design tool (crispr.mit.edu). Among 32 possible targeting guide RNAs, “Fm_ex1_(GATATCCACACCCATGGTG)” was selected as a guide RNA to avoid off-target possibilities. Among 98 possible target sides of Fm_ex_1, based on the CRISPR Design tool, there were only three off-targets within the exonic region outside of SPATA31 genes with very low targeting score (less than 0.2). Complementary oligo-nucleotides representing the target sequence were annealed and phosphorylated according to [27 (link)]. The ligation reaction was treated for 30 min with PlasmidSafe exonuclease (Epicenter) to prevent unwanted recombination products after the ligation reaction (Quick ligation kit (NEB)) of purified vector and phosphorylated and annealed oligos. 5 μL of the ligation reaction was transformed to Top10 or Stbl3 competent cells (Life Technologies; GIBCO). Positive clones were verified by sequencing and DNA was isolated using the Maxiprep plasmid DNA preparation kit (Qiagen).
6
Culturing Mammalian and Bacterial Cell Lines
7
Culturing Streptococcus pyogenes and Escherichia coli
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S. pyogenes strains were selected from the stock culture collection of the Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry (Osaka, Japan) and Institute of Medical Microbiology, Virology and Hygiene, University of Rostock (Rostock, Germany) (Köller et al.,2010 ; Murakami et al., 2002 ). A serotype M49 strain 591 was isolated from a subject with skin infection. S. pyogenes clinical isolates and mutant strains were cultured in Todd‐Hewitt broth (Becton Dickinson) supplemented with 0.2% yeast extract (Becton Dickinson) (THY medium) at ≤37°C in an ambient atmosphere. The E. coli strains XL10‐gold (Stratagene) and TOP10 (Life Technologies) served as hosts for derivatives of plasmids pSET4s, pAT18 and pMSP3545 (Trieu‐Cuot et al., 1991 ; Bryan et al., 2000 ; Takamatsu et al., 2001 ). E. coli strains were cultured in Luria‐Bertani (LB) medium at 37°C. L. lactis strain NZ9000 was cultured in M17 medium (Gibco) supplemented with 0.5% glucose (WAKO) (MG medium). For the selection and maintenance of mutant strains, antibiotics were used to supplement bacterial cultures at the following concentrations: ampicillin (Sigma‐Aldrich), 100 µg/ml for E. coli; spectinomycin (Wako), 100 µg/ml for both E. coli and S. pyogenes; and erythromycin (Sigma‐Aldrich), 300 µg/ml for E. coli and 1 µg/ml for L. lactis and S. pyogenes.
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S. pyogenes strains were selected from the stock culture collection of the Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry (Osaka, Japan) and Institute of Medical Microbiology, Virology and Hygiene, University of Rostock (Rostock, Germany) (Köller et al.,
8
Murine Melanoma Cell Lines: Metastatic Potential
9
Bacterial Cultivation and Transformation Protocols
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If not stated otherwise, Escherichia coli BL21(DE3) (Agilent Technologies Inc., Santa Clara, USA) and TOP10 (Life Technologies GmbH, Darmstadt, Germany) were cultivated at 37°C in LB (lysogeny broth) medium [35 (link)], A. tumefaciens GV3101 [36 (link)] and derivatives were grown at 30°C in YEB (yeast extract broth) and Xcv strain 85–10 [37 (link)] and derivatives in NYG (nutrient yeast glycerol) [38 (link)] supplemented with appropriate antibiotics. Plasmids were introduced into E. coli and A. tumefaciens by electroporation, and into Xcv by conjugation using pRK2013 as helper plasmid in triparental matings [39 (link)].
10
Bacterial Cultivation and Genetic Manipulation
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Escherichia coli BL21(DE3) (Agilent Technologies Inc., Santa Clara, USA) and TOP10 (Life Technologies GmbH, Darmstadt, Germany) were cultivated at 37 °C in LB (lysogeny broth) medium41 (link), A. tumefaciens GV310142 (link) and derivatives at 30 °C in YEB (yeast extract broth) and Xcv strain 85-103 and derivatives on NYG (nutrient yeast glycerol) agar plates43 (link) supplemented with appropriate antibiotics. Plasmids were introduced into E. coli and A. tumefaciens by electroporation, and into Xcv by conjugation using pRK2013 as helper plasmid in triparental matings44 (link).
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Escherichia coli BL21(DE3) (Agilent Technologies Inc., Santa Clara, USA) and TOP10 (Life Technologies GmbH, Darmstadt, Germany) were cultivated at 37 °C in LB (lysogeny broth) medium41 (link), A. tumefaciens GV310142 (link) and derivatives at 30 °C in YEB (yeast extract broth) and Xcv strain 85-103 and derivatives on NYG (nutrient yeast glycerol) agar plates43 (link) supplemented with appropriate antibiotics. Plasmids were introduced into E. coli and A. tumefaciens by electroporation, and into Xcv by conjugation using pRK2013 as helper plasmid in triparental matings44 (link).
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