SgRNA library, knock-down gene library and construction method and application of knock-down gene library

Abstract

The invention relates to the technical field of bioengineering, and particularly provides an sgRNA library, a knock-down gene library and a construction method and application of the knock-down gene library. The sgRNA library comprises sequences as shown in SEQ ID NO. 1 to 1740. The construction method comprises the following steps: constructing an MFD knock-down plasmid library by using the sgRNA library, an enzyme digestion plasmid, a T4DNA ligase, a pir E.coli competent cell, an MFD pir E.coli competent cell and the like, and transferring the MFD knock-down plasmid library into klebsiella pneumoniae by using a Tn7 transposon system through bacterial conjugation, and inserting the MFD knock-down plasmid library into a genome for stable inheritance. According to the method, the large-scale knock-down gene library can be quickly and efficiently constructed, the limitation that essential genes cannot be knocked out, and knock-out deletion, insertion mutation and the like exist is avoided, and the obtained knock-down gene library can be used for screening potential drug action targets of klebsiella pneumoniae.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a sgRNA library, a knockdown gene library, and a construction method and application of the knockdown gene library. Background technique [0002] The drug resistance of bacteria poses a serious threat to human health and has developed into a global public health safety issue, which makes the research and development of new antimicrobial drugs very important. However, the targets of most antibacterial drugs are the products encoded by essential bacterial genes (such as protein and RNA, etc.), which will increase the difficulty of research and development of new drugs, making the research and development of new antibacterial drugs seriously lagging behind. [0003] At present, it is generally necessary to study essential genes to verify their use as drug targets, and then develop new antibacterial drugs. The current main research methods include transposon rando...

AI Technical Summary

Problems solved by technology

The current main research methods include transposon random insertion mutagenesis technology, gene promoter replacement technology, and RNA interference technology, etc. Among them, the traditional transposon random insertion mutagenesis technology can find the essential genes of bacteria, but after the essential gene knockout, bacteria Difficult to survive, so functional studies cannot be performed on it

Gene promoter replacement technology can be used to regulate the expression of specific genes, but it needs to screen genes and their promoters in the whole genome, which is technically difficult and heavy workload, which limits the wide application of this technology

RNA interference technology can be used to regulate the expression of target genes, and is also used for drug sensitivity screening tests, but it is difficult to be widely used because it cannot guarantee gene knockdown efficiency and is difficult to control off-target effects

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