Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation
Summary The probiotic strain Escherichia coli Nissle 1917 (EcN) is a common bacterial chassis in synthetic biology developments for therapeutic applications given its long track record of safe administration in humans. Chromosomal integration of the genes of interest (GOIs) in the engineered bacteri...
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| Format: | Article |
| Language: | English |
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Wiley
2022-05-01
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| Series: | Microbial Biotechnology |
| Online Access: | https://doi.org/10.1111/1751-7915.13967 |
| _version_ | 1811288606276321280 |
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| author | Elena M. Seco Luis Ángel Fernández |
| author_facet | Elena M. Seco Luis Ángel Fernández |
| author_sort | Elena M. Seco |
| collection | DOAJ |
| description | Summary The probiotic strain Escherichia coli Nissle 1917 (EcN) is a common bacterial chassis in synthetic biology developments for therapeutic applications given its long track record of safe administration in humans. Chromosomal integration of the genes of interest (GOIs) in the engineered bacterium offers significant advantages in genetic stability and to control gene dose, but common methodologies relying on the transformation of EcN are inefficient. In this work, we implement in EcN the use of bacterial conjugation in combination with markerless genome engineering to efficiently insert multiple GOIs at different loci of EcN chromosome, leaving no antibiotic resistance genes, vector sequences or scars in the modified bacterium. The resolution of cointegrants that leads to markerless insertion of the GOIs requires expression of I‐SceI endonuclease and its efficiency is enhanced by λ Red proteins. We show the potential of this strategy by integrating different genes encoding fluorescent and bioluminescent reporters (i.e. GFP, mKate2, luxCDABE) both individually and sequentially. We also demonstrate its application for gene deletions in EcN (ΔflhDC) and to replace the endogenous regulation of chromosomal locus (i.e. flhDC) by heterologous regulatory elements (e.g. tetR‐Ptet) in order to have an ectopic control of gene expression in EcN with an external inducer to alter bacterial behaviour (e.g. flagellar motility). Whole‐genome sequencing confirmed the introduction of the designed modifications without off‐target alterations in the genome. This straightforward approach accelerates the generation of multiple modifications in EcN chromosome for the generation of living bacterial therapeutics. |
| first_indexed | 2024-04-13T03:40:15Z |
| format | Article |
| id | doaj.art-6345925d4fff4ffd9c9e177be1544087 |
| institution | Directory Open Access Journal |
| issn | 1751-7915 |
| language | English |
| last_indexed | 2024-04-13T03:40:15Z |
| publishDate | 2022-05-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj.art-6345925d4fff4ffd9c9e177be15440872022-12-22T03:04:11ZengWileyMicrobial Biotechnology1751-79152022-05-011551374139110.1111/1751-7915.13967Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugationElena M. Seco0Luis Ángel Fernández1Department of Microbial Biotechnology Centro Nacional de Biotecnología Consejo Superior de Investigaciones Científicas (CNB‐CSIC) Darwin 3, Campus UAM Cantoblanco Madrid 28049 SpainDepartment of Microbial Biotechnology Centro Nacional de Biotecnología Consejo Superior de Investigaciones Científicas (CNB‐CSIC) Darwin 3, Campus UAM Cantoblanco Madrid 28049 SpainSummary The probiotic strain Escherichia coli Nissle 1917 (EcN) is a common bacterial chassis in synthetic biology developments for therapeutic applications given its long track record of safe administration in humans. Chromosomal integration of the genes of interest (GOIs) in the engineered bacterium offers significant advantages in genetic stability and to control gene dose, but common methodologies relying on the transformation of EcN are inefficient. In this work, we implement in EcN the use of bacterial conjugation in combination with markerless genome engineering to efficiently insert multiple GOIs at different loci of EcN chromosome, leaving no antibiotic resistance genes, vector sequences or scars in the modified bacterium. The resolution of cointegrants that leads to markerless insertion of the GOIs requires expression of I‐SceI endonuclease and its efficiency is enhanced by λ Red proteins. We show the potential of this strategy by integrating different genes encoding fluorescent and bioluminescent reporters (i.e. GFP, mKate2, luxCDABE) both individually and sequentially. We also demonstrate its application for gene deletions in EcN (ΔflhDC) and to replace the endogenous regulation of chromosomal locus (i.e. flhDC) by heterologous regulatory elements (e.g. tetR‐Ptet) in order to have an ectopic control of gene expression in EcN with an external inducer to alter bacterial behaviour (e.g. flagellar motility). Whole‐genome sequencing confirmed the introduction of the designed modifications without off‐target alterations in the genome. This straightforward approach accelerates the generation of multiple modifications in EcN chromosome for the generation of living bacterial therapeutics.https://doi.org/10.1111/1751-7915.13967 |
| spellingShingle | Elena M. Seco Luis Ángel Fernández Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation Microbial Biotechnology |
| title | Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation |
| title_full | Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation |
| title_fullStr | Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation |
| title_full_unstemmed | Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation |
| title_short | Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation |
| title_sort | efficient markerless integration of genes in the chromosome of probiotic e coli nissle 1917 by bacterial conjugation |
| url | https://doi.org/10.1111/1751-7915.13967 |
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