Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

Olorunniji, Femi J.; Lawson-Williams, Makeba; McPherson, Arlene L.; Paget, Jane E.; Stark, W. Marshall; Rosser, Susan J.

doi:10.1093/nar/gkz936

Cited by 13 publications

(16 citation statements)

References 52 publications

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“…The first of these tools is the split intein integrase system which has already been characterized in vitro 62 . Inteins are sequences that trigger autocatalytic splicing, making it possible to reconstitute proteins from fragments expressed from two separate constructs 63 , a technique that has been used previously in plants 64 .…”

Section: Resultsmentioning

confidence: 99%

An integrase toolbox to record gene-expression during plant development

et al. 2023

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There are many open questions about the mechanisms that coordinate the dynamic, multicellular behaviors required for organogenesis. Synthetic circuits that can record in vivo signaling networks have been critical in elucidating animal development. Here, we report on the transfer of this technology to plants using orthogonal serine integrases to mediate site-specific and irreversible DNA recombination visualized by switching between fluorescent reporters. When combined with promoters expressed during lateral root initiation, integrases amplify reporter signal and permanently mark all descendants. In addition, we present a suite of methods to tune the threshold for integrase switching, including: RNA/protein degradation tags, a nuclear localization signal, and a split-intein system. These tools improve the robustness of integrase-mediated switching with different promoters and the stability of switching behavior over multiple generations. Although each promoter requires tuning for optimal performance, this integrase toolbox can be used to build history-dependent circuits to decode the order of expression during organogenesis in many contexts.

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Section: Resultsmentioning

confidence: 99%

An integrase toolbox to record gene-expression during plant development

et al. 2023

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“…Identification and characterization of more LSI RDFs will also enhance the flexibility of these systems for use in precision and programmable DNA rearrangements. [8,9,22,26,61,62] AUTHOR CONTRIBUTIONS…”

Section: Discussionmentioning

confidence: 99%

“…The reaction products were separated by agarose gel electrophoresis, then stained with SYBR safe and visualized as previously described, using a BioRad GelDoc apparatus. [21,22] The intensities of DNA bands on the gel were quantitated with the volume analysis tool of Image Lab software (BioRad) using automatic band detection. These were manually corrected to exclude artifact bands.…”

Section: In Vitro Recombination Of Supercoiled Plasmid Substrates And...mentioning

confidence: 99%

High fidelity one‐pot DNA assembly using orthogonal serine integrases

Abioye

Lawson-Williams

Lecanda

et al. 2022

Biotechnology Journal

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Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. Methods and Major Results:In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing. Conclusions and Implications:We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications.

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“…In order to circumvent this issue, different strategies as the use of degradation tags to regulate the half-life of the integrase or an induction system relying on logic gates to regulate expression of the operators could be implemented. In this regards, a split PhiC31 integrase that can be reconstituted after trans splicing of both components has been developed recently (48).…”

Section: Discussionmentioning

confidence: 99%

A reversible memory switch for plant synthetic biology based on the phage PhiC31 integration system

Bernabé‐Orts

Quijano‐Rubio

Mancheño-Bonillo

et al. 2019

Preprint

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Plant synthetic biology aims to contribute to global food security by engineering plants with new or improved functionalities. In recent years, synthetic biology has rapidly advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits able to provide organisms with novel functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to their complex multicellular nature and the lack of reliable DNA devices that allow an accurate design of more sophisticated biological circuits. Among these basic devices, gene switches are crucial to deploying new layers of regulation into the engineered organisms. Of special interest are bistable genetic toggle switches, which allow a living organism to exist in two alternative states and switch between them with a minimal metabolic burden. Naturally occurring toggle switches control important decision-making processes such as cell fate and developmental events. We sought to engineer whole plants with an orthogonal genetic toggle switch to be able to regulate artificial functions with minimal interference with their natural pathways. Here we report a bistable toggle memory switch for whole plants based on the phage PhiC31 serine integrase and its cognate recombination directionality factor (RDF). This genetic device was designed to control the transcription of two genes of interest by inversion of a central DNA regulatory element. Each state of the device is defined by one transcriptionally active gene of interest, while the other one remains inactive. The state of the switch can be reversibly modified by the action of the recombination actuators, which were administered externally (e.g. via agroinfiltration), or produced internally in response to an inducible chemical stimulus. We extensively characterized the kinetics, memory, and reversibility of this genetic switch in Nicotiana benthamiana through transient and stable transformation experiments using transgenic plants and hairy roots. Finally, we coupled the integrase expression to an estradiol-inducible promoter as a proof of principle of inducible activation of the switch.

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Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

Cited by 13 publications

References 52 publications

An integrase toolbox to record gene-expression during plant development

An integrase toolbox to record gene-expression during plant development

High fidelity one‐pot DNA assembly using orthogonal serine integrases

A reversible memory switch for plant synthetic biology based on the phage PhiC31 integration system

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