A microfluidic optimal experimental design platform for forward design of cell-free genetic networks

Sluijs, Bob van; Maas, Roel; Linden, Ardjan J. van der; Greef, Tom F. A. de; Huck, Wilhelm T. S.

doi:10.1038/s41467-022-31306-3

Cited by 19 publications

(34 citation statements)

References 82 publications

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“…In step one a hybrid swarm/evolutionary algorithm evolves an input ow pro le for each of the six inputs at three different owrates. 27,28 This algorithm scores input patterns by minimizing the D-Fisher information criterion (Supplementary information 1.2 Eq. 4).…”

Section: Resultsmentioning

confidence: 99%

Inverse Design of Enzymatic Reaction Network States

Sluijs

Zhou

Helwig

et al. 2023

Preprint

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Kinetic modelling of in vitro constructed enzymatic reaction works is vital to understand and control the complex behaviours emerging from the abundant nonlinear interactions inside. However, modelling is severely hampered by the lack of training data. Here, we introduce a methodology that combines an active learning-like approach and flow chemistry to efficiently create optimized datasets for a highly interconnected enzymatic reactions network with multiple inputs and multiple outputs. The optimal experimental design (OED) algorithm designed a sequence of out-of-equilibrium perturbations to maximise the information about the reaction kinetics, yielding a descriptive model that allowed inverse design of the output of the network towards any cost function. We experimentally validated the model by forcing the network to produce different product ratios while maintaining a minimum level of overall conversion efficiency. Our workflow scales with the complexity of the system and enables the inverse design of previously unobtainable network outputs.

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Section: Resultsmentioning

confidence: 99%

Inverse Design of Enzymatic Reaction Network States

Sluijs

Zhou

Helwig

et al. 2023

Preprint

Self Cite

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“…10). Therefore, an iterative approach was implemented, by repeatedly segmenting cells using different global image filters and segmentation parameters 43 (SN2 Fig. 9 and 13).…”

Section: Resultsmentioning

confidence: 99%

ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

Sluijs¹,

Blay²,

Stepanov³

et al. 2022

Preprint

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Cell fate decisions are ubiquitous and play a critical role throughout development, yet how localization impacts cellular decision making remains unclear. To identify the drivers of position-dependent fate decisions at a molecular level, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridization (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins and phosphoproteins in single cells at sub-micrometre spatial resolution. We studied 67 unique (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns, yielding quantification of both abundance and localization of mRNAs and (phospho-)proteins during the first 48 hours of differentiation. ARTseq-FISH revealed a fate decision between continued self-renewal and differentiation that relies solely on the position of each mESC on the micropattern. Our results demonstrate that temporal changes in cell cycle orchestrate these position-dependent cell fate decisions.

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“…An IVTT reaction is composed of three main elements: lysate, feeding buffer, and DNA templates. The lysate and feeding buffer were previously described . In this work, however, E.…”

Section: Methodsmentioning

confidence: 99%

Noise Minimization in Cell-Free Gene Expression

Bartelds,

García-Blay,

Verhagen

et al. 2023

ACS Synth. Biol.

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Biochemical reactions that involve small numbers of molecules are accompanied by a degree of inherent randomness that results in noisy reaction outcomes. In synthetic biology, the ability to minimize noise particularly during the reconstitution of future synthetic protocells is an outstanding challenge to secure robust and reproducible behavior. Here we show that by encapsulation of a bacterial cell-free gene expression system in water-in-oil droplets, in vitro-synthesized MazF reduces cell-free gene expression noise >2-fold. With stochastic simulations we identify that this noise minimization acts through both increased degradation and the autoregulatory feedback of MazF. Specifically, we find that the expression of MazF enhances the degradation rate of mRNA up to 18-fold in a sequence-dependent manner. This sequence specificity of MazF would allow targeted noise control, making it ideal to integrate into synthetic gene networks. Therefore, including MazF production in synthetic biology can significantly minimize gene expression noise, impacting future design principles of more complex cell-free gene circuits.

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A microfluidic optimal experimental design platform for forward design of cell-free genetic networks

Cited by 19 publications

References 82 publications

Inverse Design of Enzymatic Reaction Network States

Inverse Design of Enzymatic Reaction Network States

ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

Noise Minimization in Cell-Free Gene Expression

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