二元载体拷贝数工程改进了农杆菌介导的转化

IF 33.1 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Matthew J. Szarzanowicz, Lucas M. Waldburger, Michael Busche, Gina M. Geiselman, Liam D. Kirkpatrick, Alexander J. Kehl, Claudine Tahmin, Rita C. Kuo, Joshua McCauley, Hamreet Pannu, Ruoming Cui, Shuying Liu, Nathan J. Hillson, Jacob O. Brunkard, Jay D. Keasling, John M. Gladden, Mitchell G. Thompson, Patrick M. Shih
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引用次数: 0

摘要

质粒的拷贝数与质粒的功能有关,但很少有人尝试优化拷贝数较高的突变体,以便在不同宿主的不同复制起源中使用。我们利用高通量生长耦合选择试验和定向进化方法来快速鉴定影响拷贝数的复制起源突变,并筛选出能提高农杆菌介导转化(AMT)效率的突变体。通过在用于 AMT 的质粒骨架中的二元载体中引入这些突变,我们观察到在四个不同的测试起源(pVS1、RK2、pSa 和 BBR1)中,烟曲霉的瞬时转化得到了改善。对于表现最好的来源 pVS1,我们分离出了拷贝数更高的变体,它们在拟南芥中的稳定转化效率提高了 60-100%,在油脂酵母 Rhodosporidium toruloides 中提高了 390%。我们的工作提供了一个易于部署的框架来生成质粒拷贝数变体,除了提高 AMT 效率外,还能使原核生物基因工程更加精确。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Binary vector copy number engineering improves Agrobacterium-mediated transformation

Binary vector copy number engineering improves Agrobacterium-mediated transformation

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60–100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

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来源期刊
Nature biotechnology
Nature biotechnology 工程技术-生物工程与应用微生物
CiteScore
63.00
自引率
1.70%
发文量
382
审稿时长
3 months
期刊介绍: Nature Biotechnology is a monthly journal that focuses on the science and business of biotechnology. It covers a wide range of topics including technology/methodology advancements in the biological, biomedical, agricultural, and environmental sciences. The journal also explores the commercial, political, ethical, legal, and societal aspects of this research. The journal serves researchers by providing peer-reviewed research papers in the field of biotechnology. It also serves the business community by delivering news about research developments. This approach ensures that both the scientific and business communities are well-informed and able to stay up-to-date on the latest advancements and opportunities in the field. Some key areas of interest in which the journal actively seeks research papers include molecular engineering of nucleic acids and proteins, molecular therapy, large-scale biology, computational biology, regenerative medicine, imaging technology, analytical biotechnology, applied immunology, food and agricultural biotechnology, and environmental biotechnology. In summary, Nature Biotechnology is a comprehensive journal that covers both the scientific and business aspects of biotechnology. It strives to provide researchers with valuable research papers and news while also delivering important scientific advancements to the business community.
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