Efficient De Novo Assembly of 100 kb-Scale Human Functional Immunoglobulin Heavy Variable (IGHV) Gene Fragments In Vitro.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-03-26 DOI:10.1021/acssynbio.5c00011

Haiqiong Li, Shuyao Xu, Yurui Liu, Yongqi Lu, Yunshan Ning

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引用次数: 0

Abstract

Synthetic biology provides a powerful approach to functional studies of viral and microbial genomes. However, in vitro, efficient and scarless DNA manipulation on large and complex human genomes remains an inevitable challenge. Here, we de novo design and successfully assemble human functional immunoglobulin heavy variable (IGHV) gene fragments up to hundred-kilobase (kb)-sized, using an iterative in vitro assembly via Escherichia coli (E. coli) based on Gibson isothermal assembly. We describe an efficient method for "scarless" (without leaving any non-native sequences) engineering of the assembled ordered functional IGHV gene fragments, which contain complex and highly repetitive regions. Our method provides a suitable way to construct bacterial artificial chromosomes (BACs) (30-100 kb) with common materials, easy manipulations, and low cost. The construction of ordered functional IGHV gene BACs expands the synthetic biologist's chassis repertoire. It is essential for the adaptive immune response and constructing immunity humanized animal models.

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.