A tightly regulated inducible expression system utilizing the <i>fim</i> inversion recombination switch

Ham, Timothy S.; Lee, Sung Kuk; Keasling, Jay D.; Arkin, Adam P.

doi:10.1002/bit.20916

Cited by 73 publications

(81 citation statements)

References 13 publications

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“…However, all in vivo DNA-based control or data storage systems implemented to date are "single-write" systems (11)(12)(13). Consequently, the amount of information such systems are able to store is linearly proportional to the number of implemented elements (for example, a "thermometer-code" counter capable of recording N events given N data storage elements) (13).…”

mentioning

confidence: 99%

Rewritable digital data storage in live cells via engineered control of recombination directionality

Bonnet

Subsoontorn²,

Endy³

2012

Proc. Natl. Acad. Sci. U.S.A.

324

350

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The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred cell division or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionase functions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD system performance arising from bidirectional recombination can be achieved and tuned by varying the synthesis and degradation rates of recombinase proteins. The serine recombinase functions used here do not require cell-specific cofactors and should be useful in extending computing and control methods to the study and engineering of many biological systems.DNA inversion | synthetic biology | genetic engineering | standard biological parts M ost engineered genetic data storage systems use auto-or cross-regulating bistable systems of transcription repressors or activators to define and hold state via continuous gene expression (1-4). Such epigenetic storage systems can be subject to evolutionary counter selection due to resource burdens placed on the host cell or spontaneous switching due to putatively stochastic fluctuations in cellular processes, including gene expression. Moreover, heterologous expression-based systems are difficult to redeploy given differences in gene regulatory mechanisms across organisms.Another approach for storing data inside organisms is to code extrinsic information within genetic material (5). Nucleic acids have undergone natural selection to serve as heritable data storage material in organismal lineages. Moreover, DNA provides attractive features in terms of data storage robustness, scalability, and stability (6). In addition, engineered transmission of DNA molecules could support data exchange between organisms as needed to implement higher-order multicellular behaviors within programmed consortia (6, 7).Practically, researchers have begun to use enzymes that modify DNA, typically site-specific recombinases, to study and control engineered genetic systems. For example, recombinases can catalyze strand exchange between specific DNA sequences and enable precise manipulation of DNA in vitro and in vivo (8). Depending on the relative location or...

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mentioning

confidence: 99%

Rewritable digital data storage in live cells via engineered control of recombination directionality

Bonnet

Subsoontorn²,

Endy³

2012

Proc. Natl. Acad. Sci. U.S.A.

324

350

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“…The advantages of modelling the fim system over a generic FABMOS system are that the assumptions can be minimized by incorporating experimental data, and the predictions can be tested experimentally (note: a general description of FABMOS is also provided in Supplementary Note 1). The fim switch has been used in synthetic circuits to prevent 'leaky' target gene expression 25 , generate multiple discrete states with other recombinase systems 26 and control chemotaxis 27 .…”

Section: Resultsmentioning

confidence: 99%

“…Bimodal distributions can also be generated by mechanisms that do not involve recombinases or feedback regulation, including DNA methylation 47 , sequestration 48 and futile cycles 49 , but how to tune the frequency and bias of switching with these mechanisms to implement FABMOS is unclear. Other advantages of the fim system include not requiring a specialized host or conditions 25 , that high FimB and FimE concentrations are not toxic and FimB regulates the frequency of both OFF-to-ON and ON-to-OFF transitions without affecting the bias.…”

Section: Discussionmentioning

confidence: 99%

Modulating the frequency and bias of stochastic switching to control phenotypic variation

et al. 2014

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Mechanisms that control cell-to-cell variation in gene expression ('phenotypic variation') can determine a population's growth rate, robustness, adaptability and capacity for complex behaviours. Here we describe a general strategy (termed FABMOS) for tuning the phenotypic variation and mean expression of cell populations by modulating the frequency and bias of stochastic transitions between 'OFF' and 'ON' expression states of a genetic switch. We validated the strategy experimentally using a synthetic fim switch in Escherichia coli. Modulating the frequency of switching can generate a bimodal (low frequency) or a unimodal (high frequency) population distribution with the same mean expression. Modulating the bias as well as the frequency of switching can generate a spectrum of bimodal and unimodal distributions with the same mean expression. This remarkable control over phenotypic variation, which cannot be easily achieved with standard gene regulatory mechanisms, has many potential applications for synthetic biology, engineered microbial ecosystems and experimental evolution.

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“…A co-culture of these strains to produce a cellulase cocktail would, therefore, reduces the overall metabolic burden and increase the ethanol yield (Brenner et al 2008). Synthetic biology also offers superior inducible systems, such as light-inducible promoters and the fim inversion system, which are capable of providing spatiotemporal changes in gene expression (Levskaya et al 2005;Ham et al 2006). With such systems, it is possible to regulate the expression of genes with time and, potentially, to help reduce the metabolic burden imposed on the recombinant host (Drepper et al 2011).…”

Section: Future Of Cellulosic Ethanolmentioning

confidence: 99%

Heterologous Expression and Extracellular Secretion of Cellulases in Recombinant Microbes

Parisutham¹,

Lee²

2012

Bioethanol

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A tightly regulated inducible expression system utilizing the fim inversion recombination switch

Cited by 73 publications

References 13 publications

Rewritable digital data storage in live cells via engineered control of recombination directionality

Rewritable digital data storage in live cells via engineered control of recombination directionality

Modulating the frequency and bias of stochastic switching to control phenotypic variation

Heterologous Expression and Extracellular Secretion of Cellulases in Recombinant Microbes

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