Binary vector copy number engineering improves Agrobacterium-mediated transformation

Szarzanowicz, Matthew J.; Waldburger, Lucas M.; Busche, Michael; Geiselman, Gina M.; Kirkpatrick, Liam D.; Kehl, Alexander J.; Tahmin, Claudine; Kuo, Rita C.; McCauley, Joshua; Pannu, Hamreet; Cui, Ruoming; Liu, Shuying; Hillson, Nathan J.; Brunkard, Jacob O.; Keasling, Jay D.; Gladden, John M.; Thompson, Mitchell G.; Shih, Patrick M.

doi:10.1038/s41587-024-02462-2

Title: Binary vector copy number engineering improves Agrobacterium-mediated transformation

Journal Article · 03 November 2024 · Nature Biotechnology

DOI: https://doi.org/10.1038/s41587-024-02462-2 · OSTI ID:2476085

; Waldburger, Lucas M.;

; Geiselman, Gina M.;

; Kehl, Alexander J.; Tahmin, Claudine; Kuo, Rita C.;

; Pannu, Hamreet; Cui, Ruoming; Liu, Shuying;

;

; Gladden, John M.;

;

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.

View Journal Article

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); National Science Foundation (NSF); US Department of Agriculture (USDA); USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2476085

Journal Information:: Nature Biotechnology, Journal Name: Nature Biotechnology; ISSN 1087-0156

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (49)

Engineering Rhodosporidium toruloides for increased lipid production : Engineering Zhang, Shuyan; Skerker, Jeffrey M.; Rutter, Charles D. Biotechnology and Bioengineering, Vol. 113, Issue 5 https://doi.org/10.1002/bit.25864	journal	November 2015
Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity Zambryski, P.; Joos, H.; Genetello, C. The EMBO Journal, Vol. 2, Issue 12 https://doi.org/10.1002/j.1460-2075.1983.tb01715.x	journal	December 1983
Low- and intermediate-copy-number cloning vectors based on thePseudomonas plasmid pVS1 Itoh, Y.; Soldati, L.; Leisinger, T. Antonie van Leeuwenhoek, Vol. 54, Issue 6 https://doi.org/10.1007/BF00588392	journal	January 1988
Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar Zhi, Li; TeRonde, Susan; Meyer, Sandra Plant Cell Reports, Vol. 34, Issue 5 https://doi.org/10.1007/s00299-014-1734-0	journal	January 2015
Genetic tools for reliable gene expression and recombineering in Pseudomonas putida Cook, Taylor B.; Rand, Jacqueline M.; Nurani, Wasti Journal of Industrial Microbiology & Biotechnology, Vol. 45, Issue 7 https://doi.org/10.1007/s10295-017-2001-5	journal	January 2018
An improved ternary vector system for Agrobacterium-mediated rapid maize transformation Anand, Ajith; Bass, Steven H.; Wu, Emily Plant Molecular Biology, Vol. 97, Issue 1-2 https://doi.org/10.1007/s11103-018-0732-y	journal	April 2018
A simplified and efficient Agrobacterium tumefaciens electroporation method Kámán-Tóth, Evelin; Pogány, Miklós; Dankó, Tamás 3 Biotech, Vol. 8, Issue 3 https://doi.org/10.1007/s13205-018-1171-9	journal	February 2018
Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria Fang, Ferric C.; Durland, Ross H.; Helinski, Donald R. Gene, Vol. 133, Issue 1 https://doi.org/10.1016/0378-1119(93)90217-Q	journal	October 1993
Annotation of plasmid genes Thomas, Christopher M.; Thomson, Nicholas R.; Cerdeño-Tárraga, Ana M. Plasmid, Vol. 91 https://doi.org/10.1016/j.plasmid.2017.03.006	journal	May 2017
Directed evolution of copy number of a broad host range plasmid for metabolic engineering Tao, Luan; Jackson, Raymond E.; Cheng, Qiong Metabolic Engineering, Vol. 7, Issue 1 https://doi.org/10.1016/j.ymben.2004.05.006	journal	January 2005
A Suite of Constitutive Promoters for Tuning Gene Expression in Plants Zhou, Andy; Kirkpatrick, Liam D.; Ornelas, Izaiah J. ACS Synthetic Biology, Vol. 12, Issue 5, p. 1533-1545 https://doi.org/10.1021/acssynbio.3c00075	journal	April 2023
j5 DNA Assembly Design Automation Software Hillson, Nathan J.; Rosengarten, Rafael D.; Keasling, Jay D. ACS Synthetic Biology, Vol. 1, Issue 1, p. 14-21 https://doi.org/10.1021/sb2000116	journal	December 2011
Agrobacterium tumefaciens-mediated transformation of filamentous fungi de Groot, Marcel J. A.; Bundock, Paul; Hooykaas, Paul J. J. Nature Biotechnology, Vol. 16, Issue 9, p. 839-842 https://doi.org/10.1038/nbt0998-839	journal	September 1998
Enzymatic assembly of DNA molecules up to several hundred kilobases Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan Nature Methods, Vol. 6, Issue 5, p. 343-345 https://doi.org/10.1038/nmeth.1318	journal	April 2009
Fast gapped-read alignment with Bowtie 2 Langmead, Ben; Salzberg, Steven L. Nature Methods, Vol. 9, Issue 4 https://doi.org/10.1038/nmeth.1923	journal	March 2012
A reporter for noninvasively monitoring gene expression and plant transformation He, Yubing; Zhang, Tao; Sun, Hui Horticulture Research, Vol. 7, Issue 1 https://doi.org/10.1038/s41438-020-00390-1	journal	September 2020
A plasmid system with tunable copy number Rouches, Miles V.; Xu, Yasu; Cortes, Louis Brian Georges Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-31422-0	journal	July 2022
Design of orthogonal regulatory systems for modulating gene expression in plants Belcher, Michael S.; Vuu, Khanh M.; Zhou, Andy Nature Chemical Biology, Vol. 16, Issue 8 https://doi.org/10.1038/s41589-020-0547-4	journal	May 2020
Isolation and characterization of novel mutations in the pSC101 origin that increase copy number Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F. Scientific Reports, Vol. 8, Issue 1 https://doi.org/10.1038/s41598-018-20016-w	journal	January 2018
Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template Aird, Eric J.; Lovendahl, Klaus N.; St. Martin, Amber Communications Biology, Vol. 1, Issue 1 https://doi.org/10.1038/s42003-018-0054-2	journal	May 2018
The effect of temperature on Agrobacterium tumefaciens‐mediated gene transfer to plants Dillen, Willy; De Clercq, Janniek; Kapila, Jyoti The Plant Journal, Vol. 12, Issue 6 https://doi.org/10.1046/j.1365-313x.1997.12061459.x	journal	December 1997
Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana Clough, Steven J.; Bent, Andrew F. The Plant Journal, Vol. 16, Issue 6, p. 735-743 https://doi.org/10.1046/j.1365-313x.1998.00343.x	journal	December 1998
Genetic transformation of HeLa cells by Agrobacterium Kunik, T.; Tzfira, T.; Kapulnik, Y. Proceedings of the National Academy of Sciences, Vol. 98, Issue 4 https://doi.org/10.1073/pnas.98.4.1871	journal	February 2001
Trimmomatic: a flexible trimmer for Illumina sequence data Bolger, Anthony M.; Lohse, Marc; Usadel, Bjoern Bioinformatics, Vol. 30, Issue 15 https://doi.org/10.1093/bioinformatics/btu170	journal	April 2014
Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality Chaillou, Santiago; Stamou, Pinelopi-Eleftheria; Torres, Leticia L. Nucleic Acids Research, Vol. 50, Issue 16 https://doi.org/10.1093/nar/gkac682	journal	August 2022
DoriC 12.0: an updated database of replication origins in both complete and draft prokaryotic genomes Dong, Mei-Jing; Luo, Hao; Gao, Feng Nucleic Acids Research, Vol. 51, Issue D1 https://doi.org/10.1093/nar/gkac964	journal	October 2022
Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools Ham, T. S.; Dmytriv, Z.; Plahar, H. Nucleic Acids Research, Vol. 40, Issue 18 https://doi.org/10.1093/nar/gks531	journal	June 2012
Small, Stable Shuttle Vectors Based on the Minimal pVS1 Replicon for Use in Gram-Negative, Plant-Associated Bacteria Heeb, Stephan; Itoh, Yoshifumi; Nishijyo, Takayuki Molecular Plant-Microbe Interactions, Vol. 13, Issue 2 https://doi.org/10.1094/MPMI.2000.13.2.232	journal	February 2000
Genetically refactored Agrobacterium-mediated transformation Thompson, Mitchell G.; Kirkpatrick, Liam D.; Geiselman, Gina M. https://doi.org/10.1101/2023.10.13.561914	preprint	October 2023
T-DNA Binary Vectors and Systems Lee, Lan-Ying; Gelvin, Stanton B. Plant Physiology, Vol. 146, Issue 2 https://doi.org/10.1104/pp.107.113001	journal	February 2008
Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome Oltmanns, Heiko; Frame, Bronwyn; Lee, Lan-Ying Plant Physiology, Vol. 152, Issue 3 https://doi.org/10.1104/pp.109.148585	journal	December 2009
Negative regulation of pPS10 plasmid replication: origin pairing by zipping‐up DNA‐bound RepA monomers Gasset‐Rosa, Fátima; Díaz‐López, Teresa; Lurz, Rudi Molecular Microbiology, Vol. 68, Issue 3 https://doi.org/10.1111/j.1365-2958.2008.06166.x	journal	February 2008
A contemporary reassessment of the enhanced transient expression system based on the tombusviral silencing suppressor protein P19 Jay, Florence; Brioudes, Florian; Voinnet, Olivier The Plant Journal, Vol. 113, Issue 1 https://doi.org/10.1111/tpj.16032	journal	December 2022
Replication and Control of Circular Bacterial Plasmids del Solar, Gloria; Giraldo, Rafael; Ruiz-Echevarría, María Jesús Microbiology and Molecular Biology Reviews, Vol. 62, Issue 2 https://doi.org/10.1128/MMBR.62.2.434-464.1998	journal	June 1998
Agrobacterium-Mediated Plant Transformation: the Biology behind the "Gene-Jockeying" Tool Gelvin, S. B. Microbiology and Molecular Biology Reviews, Vol. 67, Issue 1, p. 16-37 https://doi.org/10.1128/MMBR.67.1.16-37.2003	journal	March 2003
Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens Winans, S. C.; Kerstetter, R. A.; Nester, E. W. Journal of Bacteriology, Vol. 170, Issue 9 https://doi.org/10.1128/jb.170.9.4047-4054.1988	journal	September 1988
Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers. Durland, R. H.; Toukdarian, A.; Fang, F. Journal of Bacteriology, Vol. 172, Issue 7 https://doi.org/10.1128/jb.172.7.3859-3867.1990	journal	January 1990
Transcriptome architecture of the three main lineages of agrobacteria Waldburger, Lucas; Thompson, Mitchell G.; Weisberg, Alexandra J. mSystems https://doi.org/10.1128/msystems.00333-23	journal	July 2023
The pGinger Family of Expression Plasmids Pearson, Allison N.; Thompson, Mitchell G.; Kirkpatrick, Liam D. Microbiology Spectrum, Vol. 11, Issue 3 https://doi.org/10.1128/spectrum.00373-23	journal	June 2023
BglBrick vectors and datasheets: A synthetic biology platform for gene expression Lee, Taek; Krupa, Rachel A.; Zhang, Fuzhong Journal of Biological Engineering, Vol. 5, Issue 1 https://doi.org/10.1186/1754-1611-5-12	journal	January 2011
DeviceEditor visual biological CAD canvas Chen, Joanna; Densmore, Douglas; Ham, Timothy S. Journal of Biological Engineering, Vol. 6, Issue 1 https://doi.org/10.1186/1754-1611-6-1	journal	December 2012
Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks Geiselman, Gina M.; Kirby, James; Landera, Alexander Microbial Cell Factories, Vol. 19, Issue 1 https://doi.org/10.1186/s12934-020-01456-4	journal	November 2020
Agrobacterium -Mediated Plant Transformation: Biology and Applications Hwang, Hau-Hsuan; Yu, Manda; Lai, Erh-Min The Arabidopsis Book, Vol. 15 https://doi.org/10.1199/tab.0186	journal	January 2017
A One Pot, One Step, Precision Cloning Method with High Throughput Capability Engler, Carola; Kandzia, Romy; Marillonnet, Sylvestre PLoS ONE, Vol. 3, Issue 11, Article No. e3647 https://doi.org/10.1371/journal.pone.0003647	journal	November 2008
RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants Vaghchhipawala, Zarir; Radke, Sharon; Nagy, Ervin PLOS ONE, Vol. 13, Issue 11 https://doi.org/10.1371/journal.pone.0200972	journal	November 2018
Characterization of copy number mutants of plasmid pSC101. Wadood, Abdul; Dohmoto, Mitsuko; Sugiura, Shigeki The Journal of General and Applied Microbiology, Vol. 43, Issue 6 https://doi.org/10.2323/jgam.43.309	journal	January 1997
Homology-Directed Repair of a Defective Glabrous Gene in Arabidopsis With Cas9-Based Gene Targeting Hahn, Florian; Eisenhut, Marion; Mantegazza, Otho Frontiers in Plant Science, Vol. 9 https://doi.org/10.3389/fpls.2018.00424	journal	April 2018
Agrobacterium tumefaciens : A Bacterium Primed for Synthetic Biology Thompson, Mitchell G.; Moore, William M.; Hummel, Niklas F. C. BioDesign Research, Vol. 2020 https://doi.org/10.34133/2020/8189219	journal	January 2020
Review: Plant Binary Vectors of Ti Plasmid in Agrobacterium tumefaciens with a Broad Host-Range Replicon of pRK2, pRi, pSa or pVS1 Murai, Norimoto American Journal of Plant Sciences, Vol. 4, Issue 4, p. 932-939 https://doi.org/10.4236/ajps.2013.44115	journal	January 2013

Similar Records

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

Journal Article · 2018 · Scientific Reports · OSTI ID:1425440

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.; +6 more

Agrobacterium-mediated transformation of lipomyces

Patent · 2018 · OSTI ID:1426818

Dai, Ziyu; Magnuson, Jon K.; Deng, Shuang; +2 more

Quantitative Dissection of Agrobacterium Virulence to Generate a Synthetic Ti Plasmid

Journal Article · 2026 · ACS Synthetic Biology · OSTI ID:3030796

Thompson, Mitchell G; Kirkpatrick, Liam D; Szarzanowicz, Matthew J; +23 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES

Title: Binary vector copy number engineering improves Agrobacterium-mediated transformation

Citation Formats

References (49)

Similar Records

Related Subjects