Journal: BMC Biotechnology

Article Title: Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex

doi: 10.1186/s12896-018-0498-y

Figure Lengend Snippet: Intracellularly expressed Cas9 led to unexpected insertion and deletion in ura5. The inserted oligonucleotides were labeled in blue and the direct repeats were underlined

Article Snippet: The pPdc1-Cas9 plasmid for expressing the codon-optimized Streptococcus pyogenes cas9 [ ] was obtained by assembling cas9 between the strong, constitutive pdc1 promoter and pdc1 terminator via DNA assembler using pRS424 (New England Biolabs, Beverly, MA) as a backbone plasmid [ ].

Techniques: Labeling

Journal: BMC Biotechnology

Article Title: Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex

doi: 10.1186/s12896-018-0498-y

Figure Lengend Snippet: In vitro digestion of cbh1 by Cas9 and gRNAs. M: DNA molecular mass marker; lane 1: Bcu I-linearized plasmid; lane 2–4: Bcu I-linearized plasmid incubated with Cas9 and gRNA1 (lane 2), gRNA2 (lane 3), and gRNA3 (lane 4)

Article Snippet: The pPdc1-Cas9 plasmid for expressing the codon-optimized Streptococcus pyogenes cas9 [ ] was obtained by assembling cas9 between the strong, constitutive pdc1 promoter and pdc1 terminator via DNA assembler using pRS424 (New England Biolabs, Beverly, MA) as a backbone plasmid [ ].

Techniques: In Vitro, Marker, Plasmid Preparation, Incubation

Journal: BMC Biotechnology

Article Title: Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex

doi: 10.1186/s12896-018-0498-y

Figure Lengend Snippet: cbh1 disruption in T. reesei by direct transformation of Cas9/gRNA complex and a plasmid containing the pyr4 selection marker. A: SDS-PAGE analysis of the fermentation supernatants of the transformants. M: protein molecular mass marker, lanes 1–2: TU-6; lanes 3–11: transformants (T1, T2, T3, T4, T6, T7, T8, and T9, respectively) that did not express CBH1; B: agarose gel electrophoresis of the PCR products amplifying the cbh1 locus from the transformants that did not express CBH1. M: DNA molecular mass marker; lane 1: TU-6; lane 2–6: the transformants T1, T2, T7, T8, and T9, respectively; C: Schematic diagram showing the inserted DNA fragments in the edited cbh1 locus; D: Characteristics of inserted or deleted DNA fragments. The numbers for the T. reesei genes were counted from the start codon

Article Snippet: The pPdc1-Cas9 plasmid for expressing the codon-optimized Streptococcus pyogenes cas9 [ ] was obtained by assembling cas9 between the strong, constitutive pdc1 promoter and pdc1 terminator via DNA assembler using pRS424 (New England Biolabs, Beverly, MA) as a backbone plasmid [ ].

Techniques: Transformation Assay, Plasmid Preparation, Selection, Marker, SDS Page, Agarose Gel Electrophoresis

Journal: Metabolic Engineering Communications

Article Title: Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

doi: 10.1016/j.meteno.2015.09.003

Figure Lengend Snippet: Oligonucleotides, plasmids and strains used in this study. U=2-deoxyuridine.

Article Snippet: pEN669 , Template for amplifying mlcE ( S. cerevisiae codon optimized) , From Evolva Holding SA.

Techniques: Sequencing, Amplification, Plasmid Preparation, Cloning, Expressing

Journal: Metabolic Engineering Communications

Article Title: Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

doi: 10.1016/j.meteno.2015.09.003

Figure Lengend Snippet: Subcellular localization of MlcE in S. cerevisiae: (A) strain construction summary and (B) fluorescent microscopy of the constructed strains (see for experimental details).

Article Snippet: pEN669 , Template for amplifying mlcE ( S. cerevisiae codon optimized) , From Evolva Holding SA.

Techniques: Microscopy, Construct

Journal: Metabolic Engineering Communications

Article Title: Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

doi: 10.1016/j.meteno.2015.09.003

Figure Lengend Snippet: Investigation of the potential of MlcE to confer the resistance to statins in S. cerevisiae: (A) strain construction summary and (B) susceptibility assay. Ten-fold dilution series of WT (CEN.PK 113-11C) and ARX3 strains (harboring MlcE efflux pump), starting with and OD 600 of 0.02 were prepared from overnight cultures and plated on a set of YPD agar plates containing different cytotoxic compounds. The plates were incubated at 30 °C for 3 days, after which the growth of the strains was recorded by photography. The plate in the black square represents the reference plate (no compounds added to YPD) (for experimental details see ).

Article Snippet: pEN669 , Template for amplifying mlcE ( S. cerevisiae codon optimized) , From Evolva Holding SA.

Techniques: Drug Susceptibility Assay, Incubation

Journal: Metabolic Engineering Communications

Article Title: Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

doi: 10.1016/j.meteno.2015.09.003

Figure Lengend Snippet: Aerobic maximum specific growth rates and growth efficiencies calculated as Δ (OD 600,max −OD 600, t =0 ) of S. cerevisiae strains WT (CEN.PK 113-11C) and ARX3 (harboring MlcE efflux pump) on glucose and different concentrations of activated lovastatin. In the samples with 0 mM lovastatin, an equal volume of solvent was added. Averages and standard deviations were obtained from triplicate experiments.

Article Snippet: pEN669 , Template for amplifying mlcE ( S. cerevisiae codon optimized) , From Evolva Holding SA.

Techniques: Solvent, Concentration Assay