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A memory switch for plant synthetic biology based on the phage ϕC31 integration system.

ABSTRACT: Synthetic biology has advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits to provide organisms with new functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to the lack of reliable DNA parts and devices that enable precise control over these new synthetic functions. In particular, memory switches based on DNA site-specific recombination have been the tool of choice to build long-term and stable synthetic memory in other organisms, because they enable a shift between two alternative states registering the information at the DNA level. Here we report a memory switch for whole plants based on the bacteriophage ϕC31 site-specific integrase. The switch was built as a modular device made of standard DNA parts, designed to control the transcriptional state (on or off) of two genes of interest by alternative inversion of a central DNA regulatory element. The state of the switch can be externally operated by action of the ϕC31 integrase (Int), and its recombination directionality factor (RDF). The kinetics, memory, and reversibility of the switch were extensively characterized in Nicotiana benthamiana plants.

SUBMITTER: Bernabe-Orts JM

PROVIDER: S-EPMC7102980 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A memory switch for plant synthetic biology based on the phage ϕC31 integration system.

Bernabé-Orts Joan Miquel JM Quijano-Rubio Alfredo A Vazquez-Vilar Marta M Mancheño-Bonillo Javier J Moles-Casas Victor V Selma Sara S Gianoglio Silvia S Granell Antonio A Orzaez Diego D

Nucleic acids research 20200401 6

Synthetic biology has advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits to provide organisms with new functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to the lack of reliable DNA parts and devices that enable precise control over these new synthetic functions. In particular, memory switches based on DNA site-specific recombination have been th ...[more]

PMID: 32083668

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Project description:This work presents a multi-hurdle approach that addresses antimicrobial resistance by minimizing the selective pressure of antimicrobials using a novel colicinogenic-phage system. We have created two synthetic T7 phages (T7-E1 and T7-M) by inserting the gene of colicin E1 (Cea) or colicin M (Cma) into the genome of the T7 phage, thereby adding an additional colicin-based hurdle to the T7 lytic cycle. The colicin-phages' efficacy in suppressing the outgrowth of a T7-resistant sub-population within a mixed culture of Escherichia coli was demonstrated using a challenge matrix design under planktonic and structured conditions. When T7-resistant cells were present at 1% of the total planktonic population, T7-E1 delayed the outgrowth. At 0.1% resistance, T7-M delayed resistant outgrowth, whereas T7-E1 suppressed the resistant sub-population. When T7-E1 and T7-M were combined into a triple-hurdle treatment, the T7-E1/T7-M cocktail completely suppressed a mixed planktonic population of 50% resistance cell concentrations. In structured environments, the colicin-phage treatments formed clear and confluent plaque-like zones of clearing in the mixed populations of 50% resistant cells with a lawn density of 1 × 106 CFU/mL. Reducing the lawn density to 1 × 105 CFU/mL diminished the multi-hurdle treatments' effectiveness, as demonstrated by localized zones of clearing within turbid bacterial lawns, highlighting the relationship between bacterial lawn density and phage effectiveness in structured environments. Fluctuation assays revealed persistence as the predominant mechanism for overcoming the treatments by T7-sensitive E. coli. Results indicate that T7-M treatment significantly reduces persister formation compared to WT-T7, while T7-E1 unexpectedly increases persister formation significantly. This suggests a complex relationship between antimicrobial stress and persister formation.ImportanceAntimicrobial resistance (AMR) poses a significant challenge in treating bacterial infections. To address this, we present a multi-hurdle approach that combines the power of different antimicrobials to target resistance. We have weaponized the natural predator of Escherichia coli, the T7-phage, by engineering it to produce toxins called colicins, resulting in a colicin-phage antimicrobial. This multi-hurdled approach aims to decrease resistance risk because survival requires different tactics to overcome the phage and colicin activity, thus adding a hurdle in a bacterium's pathway to resistance. In cases of pre-existing resistance, the colicin effectively controlled the sub-population resistant to the phage. When investigating the emergence of resistance, we discovered that antimicrobial persistence was the predominant survival strategy. These findings reveal an essential slice of the AMR pie by emphasizing bacterial survival tactics that are not based on resistance genes. By expanding our AMR lens to include persistence, we can more effectively address treatment failure.

Dataset Information

A memory switch for plant synthetic biology based on the phage ϕC31 integration system.

Publications

A memory switch for plant synthetic biology based on the phage ϕC31 integration system.

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