The genes used for the construction of the yeast strains were prepared as described in the Supplementary Information. The plasmids, primers, and DNA fragments used are listed in Tables S1 , S2 , and S3 , respectively. Escherichia coli NovaBlue (Merck Millipore, Darmstadt, Germany) was used for plasmid preparation using the Orthodox method.
Escherichia coli novablue
Manufactured by Merck Group
Sourced in Germany
Escherichia coli NovaBlue is a competent bacterial strain commonly used in molecular biology laboratories. It is designed for high-efficiency transformation of plasmid DNA. The strain exhibits a high plasmid transformation efficiency, making it suitable for general cloning and expression experiments.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by BD (1 mentions)
Yeast nitrogen base without amino acids, by BD (1 mentions)
Peptone, by BD (1 mentions)
Zeocin, by Nacalai Tesque (1 mentions)
Hygromycin, by Nacalai Tesque (1 mentions)
Yeast extract, by Nacalai Tesque (1 mentions)
Tryptone, by Nacalai Tesque (1 mentions)
Clonnat, by Jena Biosciences (1 mentions)
2 protocols using escherichia coli novablue
1
Yeast Strain Construction Using Plasmids
2
Plasmid Construction and Yeast Cultivation
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Escherichia coli NovaBlue (Merck Millipore, Darmstadt, Germany) was used for plasmid construction and amplification. The microorganisms were routinely cultured at 37 °C and 200 rpm in LB medium [10 g/L tryptone (Nacalai Tesque, Kyoto, Japan), 5
g/L yeast extract (Nacalai Tesque), and 5 g/L NaCl] supplemented with 100 μg/mL ampicillin.
The yeast strains used in this study are listed in Table 1. P. pastoris CBS7435 Δdnl4 Δhis4 was used as a parent strain since it has improved gene targeting efficiency for homologous recombination 37 (link) . P. pastoris strains were cultivated in YPG medium [10 g/L yeast extract, 20 g/L peptone (BD Biosciences, San Jose, CA, USA), and 20 g/L glycerol], YPD medium [10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose], or SD medium [6.7 g/L yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), and 20 g/L glucose] supplemented with 20 mg/L histidine and appropriate antibiotics including 500 μg/mL G418 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 300 μg/mL hygromycin (Nacalai Tesque), 100 μg/mL Zeocin (Nacalai Tesque), and 50 μg/mL clonNAT (Jena Bioscience, Löbstedter, Germany).
g/L yeast extract (Nacalai Tesque), and 5 g/L NaCl] supplemented with 100 μg/mL ampicillin.
The yeast strains used in this study are listed in Table 1. P. pastoris CBS7435 Δdnl4 Δhis4 was used as a parent strain since it has improved gene targeting efficiency for homologous recombination 37 (link) . P. pastoris strains were cultivated in YPG medium [10 g/L yeast extract, 20 g/L peptone (BD Biosciences, San Jose, CA, USA), and 20 g/L glycerol], YPD medium [10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose], or SD medium [6.7 g/L yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), and 20 g/L glucose] supplemented with 20 mg/L histidine and appropriate antibiotics including 500 μg/mL G418 (FUJIFILM Wako Pure Chemical, Osaka, Japan), 300 μg/mL hygromycin (Nacalai Tesque), 100 μg/mL Zeocin (Nacalai Tesque), and 50 μg/mL clonNAT (Jena Bioscience, Löbstedter, Germany).
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