Linearized DNA vector pHB-1 plasmid and test kit prepared from same and used for editing bacterial genomes

Abstract

The invention discloses a linearized DNA vector pHB-1 plasmid and a test kit prepared from the same. The genetic sequence of the linearized DNA vector pHB-1 plasmid is shown as SEQ ID NO: 1; the testkit comprises a linearized DNA vector pHB-1 plasmid, an efficient T4DNA ligase, a PCR sequencing primer tL3-seq and an experimental control substance. Wherein the experimental control comprises a specific gRNA sequence primer for editing an escherichia coli MlaC gene, a DNA repair template for introducing MlaC L11R point mutation and a PCR sequencing primer for confirming the MlaC L11R point mutation; the test kit disclosed by the invention can be used for carrying out gene editing experiments on various bacteria, has repeatability and stability, and can be used for editing one or more targetgenes, and the editing efficiency can reach 90-100%.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and belongs to the field of high-end biotechnology, in particular to a linearized DNA carrier pHB-1 plasmid and a kit for editing bacterial genomes prepared therewith. Background technique [0002] Bacterial genome editing is the purpose of studying its functions and traits by interrupting, deleting or replacing target genes on the genome. It plays an important role in gene editing and its application can be extended to engineering bacteria transformation, antibiotic research and development, and resistance Drug mechanism research and many other aspects. Although traditional plasmid transformation methods can be used to achieve transformation in a variety of bacteria, it is time-consuming and labor-intensive, and cannot precisely control the genome transformation position and transformation sequence, and large-scale sequencing and repeated induction are required for a transformed micr...

AI Technical Summary

Problems solved by technology

However, at present, no domestic company has developed a kit product for bacterial genome editing, and only companies such as Integrated DNA Technologies (IDT), New England Biolabs (NEB) and Thermo Fisher have developed related gene editing products for scientific research abroad, but most of them are concentrated in For gene editing of eukaryotic cells, there is no easy-to-use kit product for bacterial genome editing. In addition, the retail price of foreign gene editing reagents in my country is relatively high, even more than twice the price in foreign countries, and the cost performance is very low; , using commercially available reagents to prepare sgRNA necessary for gene editing or construct vectors, usually takes days or weeks

Although chemical synthesis can be used to directly synthesize sgRNA, it is expensive and not suitable for bacterial gene editing due to the widespread presence of RNA degrading enzymes in bacterial culture environments

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