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The synthetic integron: an in vivo genetic shuffling device.

Bikard D, Julié-Galau S, Cambray G, Mazel D - Nucleic Acids Res. (2010)

Bottom Line: Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering.We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities.Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Département Génomes et Génétique, Paris, France.

ABSTRACT
As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with 'regulatory' elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.

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Alternative experimental setups. (A) Integron cassettes are shuffled directly on a plasmid in a cloning strain. They are then extracted and transformed in the trp− selection strain. (B) Integron cassettes are delivered through conjugation into the trp− strain where they are recombined on the chromosome. Color code is the same as in figure one, except for cell with a different genetic background, which are colored in grey.
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Figure 3: Alternative experimental setups. (A) Integron cassettes are shuffled directly on a plasmid in a cloning strain. They are then extracted and transformed in the trp− selection strain. (B) Integron cassettes are delivered through conjugation into the trp− strain where they are recombined on the chromosome. Color code is the same as in figure one, except for cell with a different genetic background, which are colored in grey.

Mentions: Overall, these results demonstrate that a synthetic integron can efficiently be used to generate functional gene combinations from a library of independent candidate gene cassettes. In order to improve the flexibility of the system presented above, we devised and tested two alternative methods that allow one to carry out the rearrangement and selection steps in different genetic backgrounds and enhance the delivery of cassettes. Having the synthetic integron on the chromosome presents some disadvantages in those cases one wants to generate combinations in one strain and select for good solutions in another genetic background. To address this concern, we assessed the potential of a synthetic integron carried by a plasmid (Figure 3B). The original cassette array was cloned into a low copy number plasmid [BBa_pSB4C5 (23)] and recombination was induced during an overnight culture. Plasmid DNA was recovered and transformed into a tryptophan auxotroph, and transformants where plated on minimal medium to select for plasmids carrying functional trp operons. The proportion of prototrophs among the transformants was 2.2 × 10−4. Subsequent analysis revealed that most of them contained multiple plasmids each carrying different genes of the tryptophan pathway. Nevertheless, out of the 96 colonies screened, we were able to identify six clones carrying all the genes in a single plasmid. They were all in different combinations, and three of them carried duplications of one gene or more (see Supplementary Table S1).Figure 3.


The synthetic integron: an in vivo genetic shuffling device.

Bikard D, Julié-Galau S, Cambray G, Mazel D - Nucleic Acids Res. (2010)

Alternative experimental setups. (A) Integron cassettes are shuffled directly on a plasmid in a cloning strain. They are then extracted and transformed in the trp− selection strain. (B) Integron cassettes are delivered through conjugation into the trp− strain where they are recombined on the chromosome. Color code is the same as in figure one, except for cell with a different genetic background, which are colored in grey.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926619&req=5

Figure 3: Alternative experimental setups. (A) Integron cassettes are shuffled directly on a plasmid in a cloning strain. They are then extracted and transformed in the trp− selection strain. (B) Integron cassettes are delivered through conjugation into the trp− strain where they are recombined on the chromosome. Color code is the same as in figure one, except for cell with a different genetic background, which are colored in grey.
Mentions: Overall, these results demonstrate that a synthetic integron can efficiently be used to generate functional gene combinations from a library of independent candidate gene cassettes. In order to improve the flexibility of the system presented above, we devised and tested two alternative methods that allow one to carry out the rearrangement and selection steps in different genetic backgrounds and enhance the delivery of cassettes. Having the synthetic integron on the chromosome presents some disadvantages in those cases one wants to generate combinations in one strain and select for good solutions in another genetic background. To address this concern, we assessed the potential of a synthetic integron carried by a plasmid (Figure 3B). The original cassette array was cloned into a low copy number plasmid [BBa_pSB4C5 (23)] and recombination was induced during an overnight culture. Plasmid DNA was recovered and transformed into a tryptophan auxotroph, and transformants where plated on minimal medium to select for plasmids carrying functional trp operons. The proportion of prototrophs among the transformants was 2.2 × 10−4. Subsequent analysis revealed that most of them contained multiple plasmids each carrying different genes of the tryptophan pathway. Nevertheless, out of the 96 colonies screened, we were able to identify six clones carrying all the genes in a single plasmid. They were all in different combinations, and three of them carried duplications of one gene or more (see Supplementary Table S1).Figure 3.

Bottom Line: Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering.We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities.Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Département Génomes et Génétique, Paris, France.

ABSTRACT
As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with 'regulatory' elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.

Show MeSH
Related in: MedlinePlus