inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes
Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always off...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-01-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/10/2/236 |
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| author | Ranti Dev Shukla Ágnes Zvara Ákos Avramucz Alona Yu. Biketova Akos Nyerges László G. Puskás Tamás Fehér |
| author_facet | Ranti Dev Shukla Ágnes Zvara Ákos Avramucz Alona Yu. Biketova Akos Nyerges László G. Puskás Tamás Fehér |
| author_sort | Ranti Dev Shukla |
| collection | DOAJ |
| description | Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always offered a viable alternative; however, to date, it has been of limited use due to its tedious nature and often being limited to a single copy. We introduce here a strategy that uses bacterial insertion sequences, which are the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy number amplification is achieved using controlled copy-paste transposition. We display the successful use of IS<i>1</i> and IS<i>3</i> for this purpose in <i>Escherichia coli</i> cells using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements, we believe that with proper design, this strategy can be adapted to numerous other bacterial species. |
| first_indexed | 2024-03-09T21:25:13Z |
| format | Article |
| id | doaj.art-fcf5d7287c2d42708fd396b8ea17da26 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T21:25:13Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-fcf5d7287c2d42708fd396b8ea17da262023-11-23T21:12:58ZengMDPI AGMicroorganisms2076-26072022-01-0110223610.3390/microorganisms10020236inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial TransgenesRanti Dev Shukla0Ágnes Zvara1Ákos Avramucz2Alona Yu. Biketova3Akos Nyerges4László G. Puskás5Tamás Fehér6Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Eötvös Lóránd Research Network, H-6726 Szeged, HungaryLaboratory for Functional Genomics, Biological Research Centre of the Eötvös Lóránd Research Network, H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Eötvös Lóránd Research Network, H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Eötvös Lóránd Research Network, H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Eötvös Lóránd Research Network, H-6726 Szeged, HungaryLaboratory for Functional Genomics, Biological Research Centre of the Eötvös Lóránd Research Network, H-6726 Szeged, HungarySynthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Eötvös Lóránd Research Network, H-6726 Szeged, HungaryCloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always offered a viable alternative; however, to date, it has been of limited use due to its tedious nature and often being limited to a single copy. We introduce here a strategy that uses bacterial insertion sequences, which are the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy number amplification is achieved using controlled copy-paste transposition. We display the successful use of IS<i>1</i> and IS<i>3</i> for this purpose in <i>Escherichia coli</i> cells using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements, we believe that with proper design, this strategy can be adapted to numerous other bacterial species.https://www.mdpi.com/2076-2607/10/2/236chromosomal gene cloninggenome editingtransgene expressionlambda-Red-mediated recombinationinsertion sequencemobile element |
| spellingShingle | Ranti Dev Shukla Ágnes Zvara Ákos Avramucz Alona Yu. Biketova Akos Nyerges László G. Puskás Tamás Fehér inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes Microorganisms chromosomal gene cloning genome editing transgene expression lambda-Red-mediated recombination insertion sequence mobile element |
| title | inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes |
| title_full | inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes |
| title_fullStr | inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes |
| title_full_unstemmed | inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes |
| title_short | inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes |
| title_sort | inpose a flexible toolbox for chromosomal cloning and amplification of bacterial transgenes |
| topic | chromosomal gene cloning genome editing transgene expression lambda-Red-mediated recombination insertion sequence mobile element |
| url | https://www.mdpi.com/2076-2607/10/2/236 |
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