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Serine integrase chimeras with activity in E. coli and HeLa cells.

Farruggio AP, Calos MP - Biol Open (2014)

Bottom Line: The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms.Our work is the first to demonstrate chimeric serine integrase activity.This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA.

No MeSH data available.


Related in: MedlinePlus

Summary of hybrid integrase activity results.(A) Hybrid activity in E. coli. Chimeric integrases have been grouped according to their activity and type. Active hybrids were able to perform recombination on at least one of the indicated att-site pairings, while inactive hybrids were not. Positive recombination results are indicated with a checkmark and negative results are marked with an “x”. Untested combinations are denoted with a dash (“−”) and pairings that produced weak positive results (light blue colonies) are marked with a tilde (“∼”). Hybrids that aggregated when overexpressed in E. coli were not subjected to any recombination assays. (B) Hybrid activity in HeLa. The CT{8,8} integrase TcT P3 × B3 reaction is marked with a tilde because the recombination product could only be detected via PCR.
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f07: Summary of hybrid integrase activity results.(A) Hybrid activity in E. coli. Chimeric integrases have been grouped according to their activity and type. Active hybrids were able to perform recombination on at least one of the indicated att-site pairings, while inactive hybrids were not. Positive recombination results are indicated with a checkmark and negative results are marked with an “x”. Untested combinations are denoted with a dash (“−”) and pairings that produced weak positive results (light blue colonies) are marked with a tilde (“∼”). Hybrids that aggregated when overexpressed in E. coli were not subjected to any recombination assays. (B) Hybrid activity in HeLa. The CT{8,8} integrase TcT P3 × B3 reaction is marked with a tilde because the recombination product could only be detected via PCR.

Mentions: In this study, we have demonstrated that it is possible to construct functional serine integrase hybrids. We show that they can operate in E. coli on parental and/or chimeric att-sites, and that a select few are also able to function in HeLa cells. Overall, three of the four attempted hybrid architectures yielded chimeras with at least marginal activity in E. coli (CT, TC and BC; Fig. 7A). However, only two of these hybrid enzyme classes supported full catalytic domain substitutions (CT and BC), and only one chimeric architecture was robustly active in both E.coli and HeLa (BC; Fig. 7A,B).


Serine integrase chimeras with activity in E. coli and HeLa cells.

Farruggio AP, Calos MP - Biol Open (2014)

Summary of hybrid integrase activity results.(A) Hybrid activity in E. coli. Chimeric integrases have been grouped according to their activity and type. Active hybrids were able to perform recombination on at least one of the indicated att-site pairings, while inactive hybrids were not. Positive recombination results are indicated with a checkmark and negative results are marked with an “x”. Untested combinations are denoted with a dash (“−”) and pairings that produced weak positive results (light blue colonies) are marked with a tilde (“∼”). Hybrids that aggregated when overexpressed in E. coli were not subjected to any recombination assays. (B) Hybrid activity in HeLa. The CT{8,8} integrase TcT P3 × B3 reaction is marked with a tilde because the recombination product could only be detected via PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197438&req=5

f07: Summary of hybrid integrase activity results.(A) Hybrid activity in E. coli. Chimeric integrases have been grouped according to their activity and type. Active hybrids were able to perform recombination on at least one of the indicated att-site pairings, while inactive hybrids were not. Positive recombination results are indicated with a checkmark and negative results are marked with an “x”. Untested combinations are denoted with a dash (“−”) and pairings that produced weak positive results (light blue colonies) are marked with a tilde (“∼”). Hybrids that aggregated when overexpressed in E. coli were not subjected to any recombination assays. (B) Hybrid activity in HeLa. The CT{8,8} integrase TcT P3 × B3 reaction is marked with a tilde because the recombination product could only be detected via PCR.
Mentions: In this study, we have demonstrated that it is possible to construct functional serine integrase hybrids. We show that they can operate in E. coli on parental and/or chimeric att-sites, and that a select few are also able to function in HeLa cells. Overall, three of the four attempted hybrid architectures yielded chimeras with at least marginal activity in E. coli (CT, TC and BC; Fig. 7A). However, only two of these hybrid enzyme classes supported full catalytic domain substitutions (CT and BC), and only one chimeric architecture was robustly active in both E.coli and HeLa (BC; Fig. 7A,B).

Bottom Line: The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms.Our work is the first to demonstrate chimeric serine integrase activity.This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA.

No MeSH data available.


Related in: MedlinePlus