Gene knockout is defined as a mutation that inactivates gene function. This type of mutation is very beneficial for modern techniques and classical genetic studies, like functional genomics. Previously, the technique of transposition mutagenesis has been regularly used in knockouts of bacterial genes. Laborious screens were requested to search for knockout in the gene of interest. The very first use of in vitro genetic engineering for knockout of other organisms was conventionally made to alter genes contained in plasmids or Bacterial Artificial Chromosomes(BACs). Then, these modified constructs were transferred to the organism of interest by cell culture techniques.Meanwhile,the gene knockout strategy isalso a powerful tool in industry to identify new drug targets in the microorganism of interest.
Knockout is broadly used to determine the function of targeted genes, detect the protein products, and associate with the diseases that might occur when their functions are inhibited. It can be performed using molecular techniques like site-directed mutagenesis or using selection of natural mutants，also Zinc-Finger Nuclease, PCR-based, and Lambda (λ) Red Recombination, CRISPR/Cas9 technology. The CRISPR/Cas9 system has become the most popular, owing to its ease of use and rapidity. Numorus bacteria has been genetic-editting for medical research.
Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485
The gene encoding L-lactate dehydrogenase from bacteria Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned, sequenced, and used to obtain an L-ldh deletion mutant strain (TD1) following a site-specific double-crossover event as confirmed by PCR and Southern blot. Growth rates and final cell densities were similar for strain TD1 and the wild-type grown on glucose and xylose. Lactic acid was below the limit of detection (0.3 mM) for strain TD1 on both glucose and xylose at all times tested, but was readily detected for the wild-type strain, with average final concentrations of 8.1and 1.8 mM on glucose and xylose, respectively. Elimination of lactic acid as a fermentation product was accompanied by a proportional increase in the yields of acetic acid and ethanol. The results reported here represent a step toward using metabolic engineering to develop strains of thermophilic anaerobic bacteria that do not produce organic acids, and support the methodological feasibility of this goal.
Expression Vector Construction of Cellulose Synthase gRNA With CRISPR/Cas9 Gene Knockout System of Liriodendron chinense
The CRISPR/Cas system was first discovered in bacteria and is immune to DNA from invading viruses or phages. Based on the immune mechanism of the CRISPR/Cas system, the designed CRISPR/Cas9 gene editing system has been widely used in the study of genome functions of higher plants such as Arabidopsis and rice, and has become the main tool for eukaryotic gene editing. Escherichia coli used Li cellulose synthase (CESA) as the research object to construct a CRISPR/Cas9 gene knockout system expressing CESA-gRNA. The gRNA primers were designed using the GN19 NGG sequence of the first exon region at the 5'end of the LcCESA1 gene exon. The researchers used p201N-Cas9 as a vector and Gibson Assembly Master Mix as a ligase to ligate CESA1-gRNA to p201N-Cas9, and constructed the p201N-Cas9-CESA1-gRNA vector through Gibson Assembly technology. Based on the sequence of the inserted vector, the researchers designed two test primers. A sequence of appropriate size was identified by PCR amplification. The sequencing results of the inserted sequence confirmed that the gRNA was correctly connected to the vector. The p201N-Cas9-gRNA vector for directed editing of CESA gene is of great significance for determining the function of Li cellulose synthase in lily.
Comparison of Protective Efficacy between Formalin‐killed and aroA Gene‐knockout Vibrio anguillarum Vaccines in Olive Flounder, Paralichthys olivaceus
Lignin is a polyphenolic heteropolymer constituting between 18 to 35% of lignocellulose and is recognized as preventative of cellulosic biofuel commercialization. Paenibacillus polymyxa CR1 was isolated from naturally degrading corn stover and shown to produce alcohols using lignin as a sole carbon source. Genome sequencing and comparative genomics of P. polymyxa CR1 identified two homologs, a Dyp-type peroxidase and a laccase, which have previously been implicated in lignin metabolism in other bacteria. Knockout mutants of the identified genes displayed no growth deficiency and P. polymyxa CR1 is incapable of metabolizing common aromatic intermediates of lignin, suggesting the bacterium employs a novel catabolic pathway. To identify genes involved in lignin metabolism, a transposon library was generated and screened for abnormal lignin growth phenotypes. The results contained within will help elucidate the genetic basis of known functions helping delineate regulatory pathways and metabolic versatility in P. polymyxa relevant to lignin metabolism.
S. G. Desai, M. L. Guerinot & L. R. Lynd.Cloning of L-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL-YS485.Original Paper[J].2004.65:600-605
Xu Huifang;Jin Leilei;Xu Xuan;Xu Menglu;Zhang Fangchao;Zheng Chen;Chen Jinhui.Expression Vector Construction of Cellulose Synthase gRNA With CRISPR/Cas9 Gene Knockout System of Liriodendron chinense.Molecular Plant Breeding[J].2017
Eastman, Alexander W. Genomic analyses of Paenibacillus polymyxa CR1, a bacterium with potential applications in biomass degradation and biofuel production.Journal of the World Aquaculture Society[J]. 2015.