Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli.
Cold Spring Harb Protoc
; 2023(8): 107946, 2023 08 01.
Article
em En
| MEDLINE
| ID: mdl-37117030
ABSTRACT
We present a protocol for the generation of a gene-deletion allelic-exchange plasmid and its recovery in Escherichia coli for the purpose of constructing an in-frame gene deletion in Staphylococcus aureus Here, we present detailed methodologies for (i) the primer design (using the S. aureus tagO gene as our specific example); (ii) PCR amplification of the required gene fragments; (iii) preparation of the cloning vector (using the S. aureus allelic-exchange vector pIMAY* as an example); (iv) the Gibson assembly cloning method; (v) introduction of the plasmid into E. coli; (vi) confirmation of the plasmid insert in E. coli by colony PCR; and, finally, (vii) confirmation of the insert by sequencing. We also consider the long-term storage of the E. coli strains containing the desired plasmid.
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1
Base de dados:
MEDLINE
Assunto principal:
Staphylococcus aureus
/
Escherichia coli
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article