Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning
Genetic engineering of complex metabolic pathways and multiple traits often requires the introduction of multiple genes. The construction of plasmids carrying multiple DNA fragments plays a vital role in these processes. In this study, the Gibson assembly and Gateway cloning combined Pyramiding Stac...
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| Format: | Article |
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Frontiers Media S.A.
2023-11-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1263715/full |
| _version_ | 1797629429715828736 |
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| author | Dongdong Zeng Dongdong Zeng Cuiyuan Jing Lin Tang Peng He Peng He Jie Zhang Jie Zhang |
| author_facet | Dongdong Zeng Dongdong Zeng Cuiyuan Jing Lin Tang Peng He Peng He Jie Zhang Jie Zhang |
| author_sort | Dongdong Zeng |
| collection | DOAJ |
| description | Genetic engineering of complex metabolic pathways and multiple traits often requires the introduction of multiple genes. The construction of plasmids carrying multiple DNA fragments plays a vital role in these processes. In this study, the Gibson assembly and Gateway cloning combined Pyramiding Stacking of Multigenes (PSM) system was developed to assemble multiple transgenes into a single T-DNA. Combining the advantages of Gibson assembly and Gateway cloning, the PSM system uses an inverted pyramid stacking route and allows fast, flexible and efficient stacking of multiple genes into a binary vector. The PSM system contains two modular designed entry vectors (each containing two different attL sites and two selectable markers) and one Gateway-compatible destination vector (containing four attR sites and two negative selection markers). The target genes are primarily assembled into the entry vectors via two parallel rounds of Gibson assembly reactions. Then, the cargos in the entry constructs are integrated into the destination vector via a single tube Gateway LR reaction. To demonstrate PSM’s capabilities, four and nine gene expression cassettes were respectively assembled into the destination vector to generate two binary expression vectors. The transgenic analysis of these constructs in Arabidopsis demonstrated the reliability of the constructs generated by PSM. Due to its flexibility, simplicity and versatility, PSM has great potential for genetic engineering, synthetic biology and the improvement of multiple traits. |
| first_indexed | 2024-03-11T10:54:13Z |
| format | Article |
| id | doaj.art-7a8c9d970dd440b9bdd2a54517e39e01 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-11T10:54:13Z |
| publishDate | 2023-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-7a8c9d970dd440b9bdd2a54517e39e012023-11-13T12:23:55ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-11-011110.3389/fbioe.2023.12637151263715Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloningDongdong Zeng0Dongdong Zeng1Cuiyuan Jing2Lin Tang3Peng He4Peng He5Jie Zhang6Jie Zhang7The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, ChinaDepartment of Clinical Medicine, Southwest Medical University, Luzhou, Sichuan, ChinaThe Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, ChinaThe Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, ChinaThe Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, ChinaDepartment of Clinical Medicine, Southwest Medical University, Luzhou, Sichuan, ChinaThe Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, ChinaDepartment of Clinical Medicine, Southwest Medical University, Luzhou, Sichuan, ChinaGenetic engineering of complex metabolic pathways and multiple traits often requires the introduction of multiple genes. The construction of plasmids carrying multiple DNA fragments plays a vital role in these processes. In this study, the Gibson assembly and Gateway cloning combined Pyramiding Stacking of Multigenes (PSM) system was developed to assemble multiple transgenes into a single T-DNA. Combining the advantages of Gibson assembly and Gateway cloning, the PSM system uses an inverted pyramid stacking route and allows fast, flexible and efficient stacking of multiple genes into a binary vector. The PSM system contains two modular designed entry vectors (each containing two different attL sites and two selectable markers) and one Gateway-compatible destination vector (containing four attR sites and two negative selection markers). The target genes are primarily assembled into the entry vectors via two parallel rounds of Gibson assembly reactions. Then, the cargos in the entry constructs are integrated into the destination vector via a single tube Gateway LR reaction. To demonstrate PSM’s capabilities, four and nine gene expression cassettes were respectively assembled into the destination vector to generate two binary expression vectors. The transgenic analysis of these constructs in Arabidopsis demonstrated the reliability of the constructs generated by PSM. Due to its flexibility, simplicity and versatility, PSM has great potential for genetic engineering, synthetic biology and the improvement of multiple traits.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1263715/fullmultigene stackingGibson assemblygateway cloningpyramiding stacking of multigenesgenetic engineeringsynthetic biology |
| spellingShingle | Dongdong Zeng Dongdong Zeng Cuiyuan Jing Lin Tang Peng He Peng He Jie Zhang Jie Zhang Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning Frontiers in Bioengineering and Biotechnology multigene stacking Gibson assembly gateway cloning pyramiding stacking of multigenes genetic engineering synthetic biology |
| title | Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning |
| title_full | Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning |
| title_fullStr | Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning |
| title_full_unstemmed | Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning |
| title_short | Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning |
| title_sort | pyramiding stacking of multigenes psm a simple flexible and efficient multigene stacking system based on gibson assembly and gateway cloning |
| topic | multigene stacking Gibson assembly gateway cloning pyramiding stacking of multigenes genetic engineering synthetic biology |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1263715/full |
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