The integration of tandem gene repeats<i>via</i>a bacterial type-II toxin-antitoxin-mediated gene amplification (ToxAmp) system and stability visualisation in<i>Saccharomyces cerevisiae</i>
Abstract Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms, like human diseases and microbial productivities of target bioproducts. Here, we develop a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats inSaccharomyces cerevisiaeand further visualise the evolutionary trajectories of gene repeats. We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification (ToxAmp) modules, and accidentally re-visited the high-level capacity of multi-fragment co-transformation inS. cerevisiae. This system delivered the multi-copy gene integration in the form of tandem gene repeats spontaneously and independently from toxin-antitoxin-mediated selection. Inducing the toxin (RelE) expressingviaa copper (II)-inducibleCUP1promoter successfully drove thein-situgene amplification of the antitoxin (RelB) module, resulting in ∼40 copies of a green fluorescence reporter (GFP) gene per copy of genome. The copy-number changes, increasing and decreasing, and stable maintenance were visualised using the GFP and blue chromoprotein AeBlue as reporters. Copy-number increasing happened spontaneously not depending on a selection pressure and was quickly enriched through toxin-antitoxin-mediated selection. In summary, the bacterial toxin-antitoxin systems provide a flexible mechanism to manipulate gene copy number in eukaryotic cells and can be exploited for synthetic biology and metabolic engineering applications.<jats:sec id="s1">Table of Contents Graphic <jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="578080v1_ufig1" position="float" orientation="portrait" /></jats:fig>.
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Preprint| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
bioRxiv.org - (2024) vom: 02. Feb. Zur Gesamtaufnahme - year:2024 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Evans, Samuel [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2024.01.30.578080 |
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| PPN (Katalog-ID): |
XBI042348412 |
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| 100 | 1 | |a Evans, Samuel |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a The integration of tandem gene repeats<i>via</i>a bacterial type-II toxin-antitoxin-mediated gene amplification (ToxAmp) system and stability visualisation in<i>Saccharomyces cerevisiae</i> |
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| 520 | |a Abstract Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms, like human diseases and microbial productivities of target bioproducts. Here, we develop a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats inSaccharomyces cerevisiaeand further visualise the evolutionary trajectories of gene repeats. We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification (ToxAmp) modules, and accidentally re-visited the high-level capacity of multi-fragment co-transformation inS. cerevisiae. This system delivered the multi-copy gene integration in the form of tandem gene repeats spontaneously and independently from toxin-antitoxin-mediated selection. Inducing the toxin (RelE) expressingviaa copper (II)-inducibleCUP1promoter successfully drove thein-situgene amplification of the antitoxin (RelB) module, resulting in ∼40 copies of a green fluorescence reporter (GFP) gene per copy of genome. The copy-number changes, increasing and decreasing, and stable maintenance were visualised using the GFP and blue chromoprotein AeBlue as reporters. Copy-number increasing happened spontaneously not depending on a selection pressure and was quickly enriched through toxin-antitoxin-mediated selection. In summary, the bacterial toxin-antitoxin systems provide a flexible mechanism to manipulate gene copy number in eukaryotic cells and can be exploited for synthetic biology and metabolic engineering applications.<jats:sec id="s1">Table of Contents Graphic <jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="578080v1_ufig1" position="float" orientation="portrait" /></jats:fig> | ||
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| 650 | 4 | |a 570 |7 (dpeaa)DE-84 | |
| 700 | 1 | |a Lu, Zeyu |4 aut | |
| 700 | 1 | |a McDonnell, Liam |4 aut | |
| 700 | 1 | |a Anderson, Will |4 aut | |
| 700 | 1 | |a Peralta, Francisco |4 aut | |
| 700 | 1 | |a Watkins, Tyson |4 aut | |
| 700 | 1 | |a Ahmed, Hafna |4 aut | |
| 700 | 1 | |a Luna-Flores, Carlos Horacio |4 aut | |
| 700 | 1 | |a Loan, Thomas |4 aut | |
| 700 | 1 | |a Navone, Laura |4 aut | |
| 700 | 1 | |a Trau, Matt |4 aut | |
| 700 | 1 | |a Scott, Colin |0 (orcid)0000-0001-6110-6982 |4 aut | |
| 700 | 1 | |a Speight, Robert |4 aut | |
| 700 | 1 | |a Vickers, Claudia E. |4 aut | |
| 700 | 1 | |a Peng, Bingyin |4 aut | |
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