An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum
Background: The construction of plasmid vectors for transgene expression in the malaria parasite, Plasmodium falciparum, presents major technical hurdles. Traditional molecular cloning by restriction and ligation often yields deletions and re-arrangements when assembling low-complexity (A + T)-rich...
| Main Authors: | , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
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BioMed Central Ltd
2014
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| Online Access: | http://hdl.handle.net/1721.1/86003 https://orcid.org/0000-0002-7779-2216 https://orcid.org/0000-0002-6250-8796 |
| _version_ | 1811077809340153856 |
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| author | Lee, Marcus CS Fidock, David A Wagner, Jeffrey Charles Goldfless, Stephen Jacob Maddur Ganesan, Suresh Niles, Jacquin |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Lee, Marcus CS Fidock, David A Wagner, Jeffrey Charles Goldfless, Stephen Jacob Maddur Ganesan, Suresh Niles, Jacquin |
| author_sort | Lee, Marcus CS |
| collection | MIT |
| description | Background:
The construction of plasmid vectors for transgene expression in the malaria parasite, Plasmodium falciparum, presents major technical hurdles. Traditional molecular cloning by restriction and ligation often yields deletions and re-arrangements when assembling low-complexity (A + T)-rich parasite DNA. Furthermore, the use of large 5'- and 3'- untranslated regions of DNA sequence (UTRs) to drive transgene transcription limits the number of expression cassettes that can be incorporated into plasmid vectors.
Methods:
To address these challenges, two high fidelity cloning strategies, namely yeast homologous recombination and the Gibson assembly method, were evaluated for constructing P. falciparum vectors. Additionally, some general rules for reliably using the viral 2A-like peptide to express multiple proteins from a single expression cassette while preserving their proper trafficking to various subcellular compartments were assessed.
Results:
Yeast homologous recombination and Gibson assembly were found to be effective strategies for successfully constructing P. falciparum plasmid vectors. Using these cloning methods, a validated family of expression vectors that provide a flexible starting point for user-specific applications was created. These vectors are also compatible with traditional cloning by restriction and ligation, and contain useful combinations of commonly used features for enhancing plasmid segregation and site-specific integration in P. falciparum. Additionally, application of a 2A-like peptide for the synthesis of multiple proteins from a single expression cassette, and some rules for combinatorially directing proteins to discrete subcellular compartments were established.
Conclusions:
A set of freely available, sequence-verified and functionally validated parts that offer greater flexibility for constructing P. falciparum vectors having expanded expression capacity is provided. |
| first_indexed | 2024-09-23T10:48:42Z |
| format | Article |
| id | mit-1721.1/86003 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T10:48:42Z |
| publishDate | 2014 |
| publisher | BioMed Central Ltd |
| record_format | dspace |
| spelling | mit-1721.1/860032022-09-27T15:08:43Z An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum Lee, Marcus CS Fidock, David A Wagner, Jeffrey Charles Goldfless, Stephen Jacob Maddur Ganesan, Suresh Niles, Jacquin Massachusetts Institute of Technology. Department of Biological Engineering Wagner, Jeffrey Charles Goldfless, Stephen Jacob Maddur Ganesan, Suresh Niles, Jacquin Background: The construction of plasmid vectors for transgene expression in the malaria parasite, Plasmodium falciparum, presents major technical hurdles. Traditional molecular cloning by restriction and ligation often yields deletions and re-arrangements when assembling low-complexity (A + T)-rich parasite DNA. Furthermore, the use of large 5'- and 3'- untranslated regions of DNA sequence (UTRs) to drive transgene transcription limits the number of expression cassettes that can be incorporated into plasmid vectors. Methods: To address these challenges, two high fidelity cloning strategies, namely yeast homologous recombination and the Gibson assembly method, were evaluated for constructing P. falciparum vectors. Additionally, some general rules for reliably using the viral 2A-like peptide to express multiple proteins from a single expression cassette while preserving their proper trafficking to various subcellular compartments were assessed. Results: Yeast homologous recombination and Gibson assembly were found to be effective strategies for successfully constructing P. falciparum plasmid vectors. Using these cloning methods, a validated family of expression vectors that provide a flexible starting point for user-specific applications was created. These vectors are also compatible with traditional cloning by restriction and ligation, and contain useful combinations of commonly used features for enhancing plasmid segregation and site-specific integration in P. falciparum. Additionally, application of a 2A-like peptide for the synthesis of multiple proteins from a single expression cassette, and some rules for combinatorially directing proteins to discrete subcellular compartments were established. Conclusions: A set of freely available, sequence-verified and functionally validated parts that offer greater flexibility for constructing P. falciparum vectors having expanded expression capacity is provided. National Institute of General Medical Sciences (U.S.) (Biotechnology Traning Grant 5-T32-GM08334) National Institute of Environmental Health Sciences (Training Grant in Toxicology 5-T32-ES007020) National Institutes of Health (U.S.) (Director's New Innovator Award 1DP20D007124) Bill & Melinda Gates Foundation (Grand Challenges Explorations initiative OPP1069759) 2014-04-03T18:37:19Z 2014-04-03T18:37:19Z 2013-10 2013-08 2014-04-02T15:16:24Z Article http://purl.org/eprint/type/JournalArticle 1475-2875 http://hdl.handle.net/1721.1/86003 Wagner, Jeffrey C et al. “An Integrated Strategy for Efficient Vector Construction and Multi-Gene Expression in Plasmodium Falciparum.” Malaria Journal 12.1 (2013): 373. https://orcid.org/0000-0002-7779-2216 https://orcid.org/0000-0002-6250-8796 en http://dx.doi.org/10.1186/1475-2875-12-373 Malaria Journal Creative Commons Attribution http://creativecommons.org/licenses/by/2.0 Jeffrey C Wagner et al.; licensee BioMed Central Ltd. application/pdf BioMed Central Ltd BioMed Central Ltd |
| spellingShingle | Lee, Marcus CS Fidock, David A Wagner, Jeffrey Charles Goldfless, Stephen Jacob Maddur Ganesan, Suresh Niles, Jacquin An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum |
| title | An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum |
| title_full | An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum |
| title_fullStr | An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum |
| title_full_unstemmed | An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum |
| title_short | An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum |
| title_sort | integrated strategy for efficient vector construction and multi gene expression in plasmodium falciparum |
| url | http://hdl.handle.net/1721.1/86003 https://orcid.org/0000-0002-7779-2216 https://orcid.org/0000-0002-6250-8796 |
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