Escherichia coli DH5α was used for cloning and vector maintenance. The host P. pastoris strain GS115 and pPIC9K plasmid were from Invitrogen (Carlsbad, CA, USA). DH5α was grown in LB medium at 37°C. P. pastoris was cultured in YPD medium (1 % yeast extract, 2 % peptone, 2 % dextrose) and BMGY medium (1 % glycerol, 1 % YE, 2 % peptone, 1.34 % yeast nitrogen base w/o amino acids and ammonium sulfate (YNB), 0.0004% biotin at pH 6.0, and 100 mM potassium phosphate buffer) or BMMY induction medium (methanol instead of glycerol). The growth conditions for P. pastoris were 28–30°C and 300 rpm shaking.
Gs115
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GS115 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It serves as a general-purpose instrument for scientific applications. The core function of the GS115 is to provide controlled environmental conditions for various experimental processes.
Automatically generated - may contain errors
Lab products found in correlation
Ppiczαa, by Thermo Fisher Scientific (3 mentions)
Ppic9, by Thermo Fisher Scientific (3 mentions)
Ppic9k, by Thermo Fisher Scientific (2 mentions)
Salicin, by Merck Group (2 mentions)
Zeocin, by Thermo Fisher Scientific (2 mentions)
Ppic9k vector, by Thermo Fisher Scientific (2 mentions)
Furfural, by Thermo Fisher Scientific (1 mentions)
Sodium formate, by Sinopharm (1 mentions)
Escherichia colitop 10, by Takara Bio (1 mentions)
Sodium acetate, by Sinopharm (1 mentions)
Potassium chloride (kcl), by Sinopharm (1 mentions)
Sodium chloride (nacl), by Sinopharm (1 mentions)
Sodium hydroxide (naoh), by Sinopharm (1 mentions)
Vanillin, by Sinopharm (1 mentions)
Escherichia coli top10f, by Thermo Fisher Scientific (1 mentions)
Restriction enzymes, by Promega (1 mentions)
Ppic3.5k, by Thermo Fisher Scientific (1 mentions)
Pao815, by Thermo Fisher Scientific (1 mentions)
Escherichia coli e coli dh5α, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
30 protocols using gs115
1
Heterologous Protein Expression in P. pastoris
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Escherichia coli DH5α was used for cloning and vector maintenance. The host P. pastoris strain GS115 and pPIC9K plasmid were from Invitrogen (Carlsbad, CA, USA). DH5α was grown in LB medium at 37°C. P. pastoris was cultured in YPD medium (1 % yeast extract, 2 % peptone, 2 % dextrose) and BMGY medium (1 % glycerol, 1 % YE, 2 % peptone, 1.34 % yeast nitrogen base w/o amino acids and ammonium sulfate (YNB), 0.0004% biotin at pH 6.0, and 100 mM potassium phosphate buffer) or BMMY induction medium (methanol instead of glycerol). The growth conditions for P. pastoris were 28–30°C and 300 rpm shaking.
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Escherichia coli DH5α was used for cloning and vector maintenance. The host P. pastoris strain GS115 and pPIC9K plasmid were from Invitrogen (Carlsbad, CA, USA). DH5α was grown in LB medium at 37°C. P. pastoris was cultured in YPD medium (1 % yeast extract, 2 % peptone, 2 % dextrose) and BMGY medium (1 % glycerol, 1 % YE, 2 % peptone, 1.34 % yeast nitrogen base w/o amino acids and ammonium sulfate (YNB), 0.0004% biotin at pH 6.0, and 100 mM potassium phosphate buffer) or BMMY induction medium (methanol instead of glycerol). The growth conditions for P. pastoris were 28–30°C and 300 rpm shaking.
2
Sourcing Chemicals for Fungal BGL1 Cloning
3
Recombinant Phytase Production in P. pastoris
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Escherichia coli TOP10F′ (Invitrogen, Carlsbad, CA, USA) cells were used for DNA manipulations; these cells were cultivated in low-salt LB medium. Bacterial plasmid selection and maintenance was performed using 25 mg/L of zeocin (Invitrogen, Carlsbad, CA, USA). The P. pastoris strain GS115 (Invitrogen, Carlsbad, CA, USA) was used as a host cell, and this strain was cultivated in YPD medium (1% yeast extract, 2% peptone and 2% glucose). Transformants of P. pastoris were selected on YPDSZ agar plates (1% yeast extract, 2% peptone, 2% glucose, 18.2% sorbitol, 2% agar and 100 mg/L of zeocin). The DNA segments encoding phytase with a Flag-Tag and the anchored glycoprotein gene GCW61 were ligated into pPICZαA (Invitrogen, Carlsbad, CA, USA).
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Escherichia coli TOP10F′ (Invitrogen, Carlsbad, CA, USA) cells were used for DNA manipulations; these cells were cultivated in low-salt LB medium. Bacterial plasmid selection and maintenance was performed using 25 mg/L of zeocin (Invitrogen, Carlsbad, CA, USA). The P. pastoris strain GS115 (Invitrogen, Carlsbad, CA, USA) was used as a host cell, and this strain was cultivated in YPD medium (1% yeast extract, 2% peptone and 2% glucose). Transformants of P. pastoris were selected on YPDSZ agar plates (1% yeast extract, 2% peptone, 2% glucose, 18.2% sorbitol, 2% agar and 100 mg/L of zeocin). The DNA segments encoding phytase with a Flag-Tag and the anchored glycoprotein gene GCW61 were ligated into pPICZαA (Invitrogen, Carlsbad, CA, USA).
4
Cloning and Expression in E. coli and P. pastoris
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Escherichia coli JM109 strain was applied as a host for cloning and manipulating manipulation. This strain was cultured at 37°C in Luria-Bertani (LB) broth medium supplied with ampicillin. The P. pastoris strain GS115 (Invitrogen, CA, USA) and the expression vector pPICZαA were used as the expression hosts. The yeast cells were grown at 30°C on Yeast Extract–Peptone–Dextrose (YPD) Medium (1% yeast extract, 2% Bacto-peptone 2% glucose) and Yeast Extract–Peptone–Dextrose- sorbitol (YPDs) Medium (1% yeast extract, 2% peptone, 2% glucose, 1 M sorbitol) supplemented with 100 μg/mL zeocin if necessary. Restriction enzymes used for cloning were bought from Promega (Madison, Wisconsin, USA) and applied following manufacturer’s recommendations.
5
Recombinant Komagataella Expressing Chymosin
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6
Recombinant P. pastoris GS115 Production
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The recombinant pPIC9K–GuHMGR plasmid was linearized by restriction enzyme SalI and mobilized by electroporation (1500 V, 25 µF, 400 Ω) into the disarmed P. pastoris GS115 (Invitrogen, USA). An aliquot (0.5 mL) of yeast peptone dextrose (YPD) medium was then added and the cells were cultured at 30 °C, 200 rpm for 1 h. An aliquot (200 µL) of the suspension was placed on minimal dextrose (MD) solid medium and cultured at 30 °C for 2 days. Single colonies were removed and incubated on minimal medium (MM) and MD solid medium simultaneously at 30 °C for 2–4 days; the colonies growing on both MM and MD media were selected.
PCR was used to check that the recombinant P. pastoris contained the GuHMGR gene. The single colonies were used as PCR template21 and primers were as follows: forward primer, 5′-TACTATTGCCAGCATTGCTGC-3′; reverse primer, 5′-GCAAATGGCATTCTGACATCC-3′. The cycling parameters were as follows: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min.
Selected recombinant P. pastoris was induced to express the GuHMGR gene using BMGY and BMMY liquid media (30 °C, 250 rpm). The supernatant from a 96 h culture was examined by 12% SDS-PAGE using Coomassie brilliant blue staining. P. pastoris containing a void vector was used as a negative control.
PCR was used to check that the recombinant P. pastoris contained the GuHMGR gene. The single colonies were used as PCR template21 and primers were as follows: forward primer, 5′-TACTATTGCCAGCATTGCTGC-3′; reverse primer, 5′-GCAAATGGCATTCTGACATCC-3′. The cycling parameters were as follows: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min.
Selected recombinant P. pastoris was induced to express the GuHMGR gene using BMGY and BMMY liquid media (30 °C, 250 rpm). The supernatant from a 96 h culture was examined by 12% SDS-PAGE using Coomassie brilliant blue staining. P. pastoris containing a void vector was used as a negative control.
7
Escherichia coli and Pichia pastoris Cloning
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Escherichia coli DH5α was used for cloning, transformation, and propagation. The host, P. pastoris strain GS115 and pPinkα-HC plasmid (7.9 kb) were obtained from Invitrogen (USA). Escherichia coli was grown in Luria and Bertani (LB, SIGMA, Germany) medium at appropriate conditions (37°C with 200 rpm shaking). P. pastoris was cultured in YPD , SIGMA, Germany (1% yeast extract (YE), 2% peptone, 2% glucose/dextrose) and buffered glycerol-complex (BMGY) (1% glycerol, 1% YE, 2% peptone, 1.34% yeast nitrogen base w/o amino acids and ammonium sulfate (YNB), 0.0004% biotin in pH = 6.0, and 100 mM potassium phosphate buffer) media and induced in buffered methanol-complex medium (BMMY) (as per BMGY except with 0.5 or 3% v/v methanol instead of the glycerol) from Invitrogen (USA). The growth conditions for P. pastoris was 28 - 30°C and 300 rpm shaking. Unless otherwise stated, standard DNA methodologies were used.
8
Yeast Strain and Culture Conditions
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P. pastoris GS115 (Invitrogen), a histidine-deficient strain, was used for transposition activity assay, construction of the Tn-seq library and CRISPR/Cas9 editing assay. Medium for P. pastoris culture used in all experiments contained abundant nitrogen source, which was beneficial to the stability of haploid state. These medium included: YPD (1% yeast extract, 2% peptone and 2% glucose); YND (0.67% yeast nitrogen base without amino acids [YNB] and 1% glucose); YNDZr (YND supplemented with bleomycin zeocin at 100 μg/ml); YNDH (YND supplemented with histidine at 50 μg/ml); EMM (Edinburgh minimal medium without Vitamin B1, and 1% glucose); YNM (0.67% YNB and 0.5% methanol). For plates with solid medium, 2% agar powder was added. The Escherichia coli strain TOP10 used for plasmid propagation was cultivated at 37 °C in LB medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl). When necessary, ampicillin, or zeocin was added into LB medium at a final concentration of 100 or 50 μg/ml, respectively. All yeast strains were cultured at 30 °C while E. coli strains were cultured at 37 °C.
9
Pichia pastoris Protein Expression
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The K. phaffii strain GS115 was obtained from Invitrogen (Massy, USA) and served as the host strain for protein expression. The Escherichia coli (E. coli) DH5α (Invitrogen) was utilized for the propagation of recombinant vectors. The plasmids pPIC9 and pPICZαA (Invitrogen) were employed for expressing the target proteins. The plasmids pPIC3.5K and pAO815 (Invitrogen) were utilized for expressing the chaperones.
10
Recombinant Bht production in K. phaffii
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The bacterial and K. phaffii GS115 strains used in this study are shown in Table 1 . Bacteria were grown at 37°C in Luria-Bertani (LB) medium with antibiotic ampicillin (100 μg/mL) (Thermo Fisher Scientific). Growth and maintenance of GS115 (Invitrogen Life Technologies, Thermo Fisher Scientific) was described previously (Dagher and Bruno-Bárcena, 2016 (link)). E. coli XL1-Blue was used as the cloning host (Agilent Technologies, Thermo Fisher Scientific). The plasmid pPIC9 (Invitrogen Life Technologies, Thermo Fisher Scientific) was used as cloning vector containing codon optimized Bht (rBht sequences) (GenBank accession number JF29828).
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