Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria

Zilberzwige‐Tal, Shai; Alon, Dan Mark; Gazit, Danielle; Zachariah, Shahar; Hollander, Amit; Gazit, Ehud; Elbaz, Johann

doi:10.1021/acssynbio.0c00586

Cited by 6 publications

(5 citation statements)

References 44 publications

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“…Expression of synthetic gene cassettes further places stress on engineered populations, increasing sensitivity to external disturbances ( 11 ). Thus, gene circuits that behave consistently in nutrient-rich growth conditions in the lab can oftentimes break down when transferred to more complex and non-traditional environments ( 12 ).…”

Section: Introductionmentioning

confidence: 99%

Host evolution improves genetic circuit function in complex growth environments

Zhang,

Lezia,

Emmanuele

et al. 2024

Preprint

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Genetically engineered bacteria have become an attractive platform for numerous biomedical and industrial applications. Despite genetic circuitry functioning predictably under favorable growth conditions in the lab, the same cannot be said when placed in more complex environments for eventual deployment. Here, we used a combination of evolutionary and rational engineering approaches to enhanceE. colifor robust genetic circuit behavior in non-traditional growth environments. We utilized adaptive laboratory evolution (ALE) onE. coliMG1655 in a minimal media with a sole carbon source and saw improved dynamics of a population-lysis-based circuit after host evolution. Additionally, we improved lysis circuit tolerance of a more clinically relevant strain, the probioticE. coliNissle, using ALE of the host strain in a more complex media environment with added reactive oxygen species (ROS) stress. We observed improved recovery from circuit-induced lysis in the evolved Nissle strain, and in combination with directed mutagenesis, recovered circuit function in the complex media. These findings serve as a proof-of-concept that relevant strains of bacteria can be optimized for improved growth and performance in complex environments using ALE and that these changes can modify and improve synthetic gene circuit function for real-world applications.

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Section: Introductionmentioning

confidence: 99%

Host evolution improves genetic circuit function in complex growth environments

Zhang,

Lezia,

Emmanuele

et al. 2024

Preprint

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“…Synthetic biology aims to address pressing global challenges including disease diagnosis and treatment, , biofuel production, − contamination detection, and biomanufacturing. ,− These are achieved by engineering biological systems with new capabilities, granting cellular control and user-defined performance. , The variety of genetic circuit functions include a genetic toggle switch, genetic counters, low- or high-frequency filters, , adders, sequential asynchronous logic circuits, and more.…”

Section: Introductionmentioning

confidence: 99%

Investigating and Modeling the Factors That Affect Genetic Circuit Performance

Zilberzwige-Tal,

Fontanarrosa,

Bychenko

et al. 2023

ACS Synth. Biol.

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Over the past 2 decades, synthetic biology has yielded ever more complex genetic circuits that are able to perform sophisticated functions in response to specific signals. Yet, genetic circuits are not immediately transferable to an outside-the-lab setting where their performance is highly compromised. We propose introducing a broader test step to the design−build−test− learn workflow to include factors that might contribute to unexpected genetic circuit performance. As a proof of concept, we have designed and evaluated a genetic circuit in various temperatures, inducer concentrations, nonsterilized soil exposure, and bacterial growth stages. We determined that the circuit's performance is dramatically altered when these factors differ from the optimal lab conditions. We observed significant changes in the time for signal detection as well as signal intensity when the genetic circuit was tested under nonoptimal lab conditions. As a learning effort, we then proceeded to generate model predictions in untested conditions, which is currently lacking in synthetic biology application design. Furthermore, broader test and learn steps uncovered a negative correlation between the time it takes for a gate to turn ON and the bacterial growth phases. As the synthetic biology discipline transitions from proof-of-concept genetic programs to appropriate and safe application implementations, more emphasis on test and learn steps (i.e., characterizing parts and circuits for a broad range of conditions) will provide missing insights on genetic circuit behavior outside the lab.

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“…For this purpose, we encapsulated a functional riboswitch inside MS2 bacteriophage virus-like particles (VLPs) (Figure and Figure S1). Utilizing the VLP as the nanocarrier (NC) of the riboswitch was found to provide three important features: (i) the structure and therefore the function of the riboswitch was retained; (ii) the VLP pores permitted the passive influx of small molecules, such as metabolites; and (iii) the VLP shell acted as a protective cage against nucleases, thus overcoming one of the most significant limitations of functional RNA applications. Furthermore, MS2 VLPs hold great potential in applying them as vaccines and as a carrier to specific cells. , …”

Section: Introductionmentioning

confidence: 99%

Engineered Riboswitch Nanocarriers as a Possible Disease-Modifying Treatment for Metabolic Disorders

et al. 2022

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Both DNA- and RNA-based nanotechnologies are remarkably useful for the engineering of molecular devices in vitro and are applied in a vast collection of applications. Yet, the ability to integrate functional nucleic acid nanostructures in applications outside of the lab requires overcoming their inherent degradation sensitivity and subsequent loss of function. Viruses are minimalistic yet sophisticated supramolecular assemblies, capable of shielding their nucleic acid content in nuclease-rich environments. Inspired by this natural ability, we engineered RNA-virus-like particles (VLPs) nanocarriers (NCs). We showed that the VLPs can function as an exceptional protective shell against nuclease-mediated degradation. We then harnessed biological recognition elements and demonstrated how engineered riboswitch NCs can act as a possible disease-modifying treatment for genetic metabolic disorders. The functional riboswitch is capable of selectively and specifically binding metabolites and preventing their self-assembly process and its downstream effects. When applying the riboswitch nanocarriers to an in vivo yeast model of adenine accumulation and self-assembly, significant inhibition of the sensitivity to adenine feeding was observed. In addition, using an amyloid-specific dye, we proved the riboswitch nanocarriers’ ability to reduce the level of intracellular amyloid-like metabolite cytotoxic structures. The potential of this RNA therapeutic technology does not apply only to metabolic disorders, as it can be easily fine-tuned to be applied to other conditions and diseases.

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Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria

Cited by 6 publications

References 44 publications

Host evolution improves genetic circuit function in complex growth environments

Host evolution improves genetic circuit function in complex growth environments

Investigating and Modeling the Factors That Affect Genetic Circuit Performance

Engineered Riboswitch Nanocarriers as a Possible Disease-Modifying Treatment for Metabolic Disorders

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