Knockout And Fitness Deficiency Of Escherichia Coli Used By CRISPR/Cas9

Conserved genes are a class of conserved and functionally important genes.The loss of conserved genes during evolution may lead to defects in fitness and even related diseases.At present,it is unknown how the fitness defects caused by the loss of conserved genes affect the environmental fitness of organisms.Based on the foregoing reasons,this project uses Escherichia coli as a model organism to explore the scientific problem of the loss of prokaryotic conserved genes on their environmental fitness.In this study,we first selected non-essential conserved genes from the genome of Escherichia coli K12 MG1655 by bioinformatics,and then used CRISPR / Cas9 and Red homologous recombination technology to knock out non-essential conserved genes in Escherichia coli to construct Gene loss library,finally through bioinformatics analysis of the Escherichia coligenes that have been knocked out successfully,to find genes related to amino acid metabolism,and to study the environmental fitness of them.The conclusions are as follows:(1)Biological information analysis of non-essential conserved genes in Escherichia coliIn this study,about 3000 bacterial proteome sequences were collected from National Center for Biotechnology Information,among which there were 31 phyla,41 classes,104 orders,219 families,and non-Escherichia coli were randomly selected as candidates from BBI.Homologous sequences,of which there are 421 conserved genes,among 421 conserved genes,there are 236 non-essential conserved genes and 185 essential conserved genes.The loss rate of these 236 non-essential conserved genes is between 4%-19% and the loss phenomenon is common among different classes and even gates,suggesting that the loss of conserved genes may be neutral.Then through Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis,it was found that most of these genes are related to anabolism,suggesting that mutation or even loss of these genes may have a potential impact on bacterial growth and development.Finally,phylogenetic analysis suggested that although these genes were conservative,there were still gene losses during bacterial evolution.(2)Knockout of non-essential conserved genes in Escherichia coliIn this study,the golden gate technology was used to organically combine the four vector fragments containing CRISPR / Cas9,Red recombinant protein,homology arm,and small guide RNA.The gel electrophoresis map from the plasmid vector and the sequencing map of the plasmid vector through the successful connection can be Seeing mutations in non-functional regions on plasmid vectors indicates that the ligation method is stable and achievable for large-scale plasmid vector ligation.From the results of multiple gene-induced knockout experiments,it can be seen that it is feasible to use the arabinose promoter to induce the translation and expression of Cas9 protein and Red recombinant protein,and the Escherichia coli after the knockout has not completely died,indicating that it is not necessary for conservatism.The gene knockout method is feasible.The above entire experimental results indicate that the large-scale construction of conservative non-essential gene knockout plasmids and experimental methods for conservative non-essential gene knockout are stable and effective.(3)Study on environmental adaptability of knockout Escherichia coliIn this study,we selected 11 genes related to amino acid metabolism from 70 knockout E.coli by Kyoto Encyclopedia of Genes and Genomes enrichment and Genomes and Gene Ontology enrichment,and cultivated them under different carbon sources and temperatures.By comparing the growth curves of E.coli under different carbon sources,it can be concluded that genes b0072 and b0073 may participate in the catalysis of glycerol kinase in the first step of glycerol into the glycolysis pathway or the dehydrogenation of ?-glycerol phosphate in the second step of the reaction;gene b2329,b3771,and b3960 may be involved in glycolysis and subsequent metabolic pathways,indicating that genes b2329,b3771,and b3960 may be important and irreplaceable genes involved in the metabolic pathway of E.coli;while other genes have different effects on E.coli under different carbon source conditions.All have different effects.By comparing the growth curves of E.coli at different temperatures,it can be concluded that the loss of gene b2414 only has a strong effect on E.coli at a temperature of 30 ° C,and has no effect on the growth of E.coli at other temperatures.In summary,this study successfully constructed 80 conservative non-essential gene knockout vectors,and successfully knocked out 70 conservative non-essential genes in E.coli K12 MG1655;studied under different carbon sources and temperatures the functions of 11 conserved non-essential genes provide important information for understanding the role of these genes in biological evolution.