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
{"title":"Binary vector copy number engineering improves Agrobacterium-mediated transformation","authors":"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","doi":"10.1038/s41587-024-02462-2","DOIUrl":null,"url":null,"abstract":"<p>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 <i>Agrobacterium</i>-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of <i>Nicotiana benthamiana</i> 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 <i>Arabidopsis thaliana</i> and 390% in the oleaginous yeast <i>Rhodosporidium toruloides</i>. 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.</p>","PeriodicalId":19084,"journal":{"name":"Nature biotechnology","volume":null,"pages":null},"PeriodicalIF":33.1000,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nature biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1038/s41587-024-02462-2","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
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.
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.
京公网安备 11010802042870号
京ICP备2023020795号-1
分享
求助内容：
应助结果提醒方式：
扫码关注我们
