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71.
Yu Wang Ye Liu Junwei Li Yi Yang Xiaomeng Ni Haijiao Cheng Teng Huang Yanmei Guo Hongwu Ma Ping Zheng Meng Wang Jibin Sun Yanhe Ma 《Biotechnology and bioengineering》2019,116(11):i-i
CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ). 相似文献
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Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications. 相似文献
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New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering. 相似文献
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Christopher Minteer Marco Morselli Margarita Meer Jian Cao Albert HigginsChen Sabine M. Lang Matteo Pellegrini Qin Yan Morgan
E. Levine 《Aging cell》2022,21(2)
Aging is associated with dramatic changes to DNA methylation (DNAm), although the causes and consequences of such alterations are unknown. Our ability to experimentally uncover mechanisms of epigenetic aging will be greatly enhanced by our ability to study and manipulate these changes using in vitro models. However, it remains unclear whether the changes elicited by cells in culture can serve as a model of what is observed in aging tissues in vivo. To test this, we serially passaged mouse embryonic fibroblasts (MEFs) and assessed changes in DNAm at each time point via reduced representation bisulfite sequencing. By developing a measure that tracked cellular aging in vitro, we tested whether it tracked physiological aging in various mouse tissues and whether anti‐aging interventions modulate this measure. Our measure, termed CultureAGE, was shown to strongly increase with age when examined in multiple tissues (liver, lung, kidney, blood, and adipose). As a control, we confirmed that the measure was not a marker of cellular senescence, suggesting that it reflects a distinct yet progressive cellular aging phenomena that can be induced in vitro. Furthermore, we demonstrated slower epigenetic aging in animals undergoing caloric restriction and a resetting of our measure in lung and kidney fibroblasts when re‐programmed to iPSCs. Enrichment and clustering analysis implicated EED and Polycomb group (PcG) factors as potentially important chromatin regulators in translational culture aging phenotypes. Overall, this study supports the concept that physiologically relevant aging changes can be induced in vitro and used to uncover mechanistic insights into epigenetic aging. 相似文献
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