| Autor: |
Nicholas M. Thomson, Chuanzhen Zhang, Eleftheria Trampari, Mark J. Pallen |
| Jazyk: |
angličtina |
| Rok vydání: |
2020 |
| Předmět: |
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| Zdroj: |
BMC Biotechnology, Vol 20, Iss 1, Pp 1-10 (2020) |
| Druh dokumentu: |
article |
| ISSN: |
1472-6750 |
| DOI: |
10.1186/s12896-020-00648-5 |
| Popis: |
Abstract Background Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps. Results We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. Conclusions Our plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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