Saccharomyces cerevisiae large-fragment gene editing method
The invention discloses a saccharomyces cerevisiae large-fragment gene editing method. The editing method comprises the following steps: (1) designing a GIM gene expression cassette, specifically, amplifying an I-SceI gene expression cassette controlled by a galactose promoter from a plasmid pGSHU,...
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| creator | ZHOU SA LI SHIQI MA WENJIAN ZHANG TENGYUE CHEN TECHANG MU JIAKANG DING CHUNYANG |
| description | The invention discloses a saccharomyces cerevisiae large-fragment gene editing method. The editing method comprises the following steps: (1) designing a GIM gene expression cassette, specifically, amplifying an I-SceI gene expression cassette controlled by a galactose promoter from a plasmid pGSHU, inserting an I-SceI recognition sequence into each of an upstream primer and a downstream primer, and enabling a homologous arm to be 52bp; (2) constructing a double-DSBs mutant strain, specifically, transforming into yeast by utilizing a PEG/LiAC method, coating on a Hyg screening plate to grow, selecting a grown monoclonal strain, extracting a genome, and verifying; and (3) designing a target gene expression cassette, specifically, connecting a target gene with a KanMX6 selection marker through overlap extension PCR, and obtaining an upstream and downstream homologous arm 68bp which is a base sequence at two ends of upstream DSB in the double DSBs. According to the saccharomyces cerevisiae large-fragment gene edi |
| format | Patent |
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The editing method comprises the following steps: (1) designing a GIM gene expression cassette, specifically, amplifying an I-SceI gene expression cassette controlled by a galactose promoter from a plasmid pGSHU, inserting an I-SceI recognition sequence into each of an upstream primer and a downstream primer, and enabling a homologous arm to be 52bp; (2) constructing a double-DSBs mutant strain, specifically, transforming into yeast by utilizing a PEG/LiAC method, coating on a Hyg screening plate to grow, selecting a grown monoclonal strain, extracting a genome, and verifying; and (3) designing a target gene expression cassette, specifically, connecting a target gene with a KanMX6 selection marker through overlap extension PCR, and obtaining an upstream and downstream homologous arm 68bp which is a base sequence at two ends of upstream DSB in the double DSBs. 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The editing method comprises the following steps: (1) designing a GIM gene expression cassette, specifically, amplifying an I-SceI gene expression cassette controlled by a galactose promoter from a plasmid pGSHU, inserting an I-SceI recognition sequence into each of an upstream primer and a downstream primer, and enabling a homologous arm to be 52bp; (2) constructing a double-DSBs mutant strain, specifically, transforming into yeast by utilizing a PEG/LiAC method, coating on a Hyg screening plate to grow, selecting a grown monoclonal strain, extracting a genome, and verifying; and (3) designing a target gene expression cassette, specifically, connecting a target gene with a KanMX6 selection marker through overlap extension PCR, and obtaining an upstream and downstream homologous arm 68bp which is a base sequence at two ends of upstream DSB in the double DSBs. 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| subjects | BEER BIOCHEMISTRY CHEMISTRY COMPOSITIONS THEREOF CULTURE MEDIA ENZYMOLOGY METALLURGY MICROBIOLOGY MICROORGANISMS OR ENZYMES MUTATION OR GENETIC ENGINEERING PROCESSES USING MICROORGANISMS PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS SPIRITS VINEGAR WINE |
| title | Saccharomyces cerevisiae large-fragment gene editing method |
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