Evolution of oil droplets in a chemorobotic platform

Gutiérrez, Juan Manuel; Hinkley, Trevor; Taylor, John; Yanev, Kliment; Cronin, Leroy

doi:10.1038/ncomms6571

Cited by 111 publications

(118 citation statements)

References 24 publications

(32 reference statements)

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“…With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been proposed (e.g., biosensors 25 or hybrids with chemical evolution 26 ) can be adapted to this method if the target protein's activity can be linked to conditional M13 phage production. However, certain applications (e.g., those involving membrane proteins) would be harder to adapt, which is why other methods such as liposome display 27 have been developed.…”

Section: Applications Of the Methodsmentioning

confidence: 99%

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

2017

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Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. these include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. a typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (pM) library expressing variants of the gene of interest (GoI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. the system has been developed for the selection of new orthogonal transcription factors (tFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage l cI tF against two synthetic bidirectional promoters. the evolved tF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. this protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. the chances of propagating nonfunctional library members because of multiple infections. A GOI with the desired characteristics upregulates gene VI expression on the AP, completing the phage life cycle. For example, a randomized TF library member that activates an artificial promoter upstream of gVI will increase its own phage production (Fig. 4a). In this way, a protein with novel desired properties can be selected after several rounds of reinfection. Applications of the methodThe method has been used to evolve a set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants, and multi-input promoter architectures, and these switches have been successfully applied in downstream synthetic gene circuits 19 . In general, the method is capable of evolving any gene-or gene circuit function-on the PM that can be linked to pVI production. This is analogous to previous uses of phageassisted continuous evolution (PACE) 22 (Fig. 4). With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been...

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Section: Applications Of the Methodsmentioning

confidence: 99%

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

2017

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“…The first physical computing system is a robot capable of chemical manipulations which we have described in previous work [18]. The scheme for the operation of the computation system is shown in figure 1.…”

Section: Resultsmentioning

confidence: 99%

“…Most of the mechanical and liquid-handling elements were designed and three-dimensionally printed using a RepRap three-dimensional printer (the thermoplastic material used was polylactic acid), which means that, if there is a need for modifications in order to introduce new technical capabilities, new elements can be easily three-dimensionally printed and attached to the robot. More information about the robot's construction and capabilities can be found elsewhere [18].…”

Section: The Robotmentioning

confidence: 99%

“…n, based on the rules of physics and chemistry. We will then control state 0 to make droplets generate different states as we have recently shown [18]. In chemical droplets it is quite easy to increase the system complexity by having built-in chemical reactions in the droplets.…”

Section: The Computationsmentioning

confidence: 99%

“…An example of this process applied to real experiments can be seen in [18], where a genetic algorithm was used to navigate a defined reaction space and optimize droplet formulations to produce a set of fitness functions. Figure 4 shows the droplet trajectories for 1 min experiments for three different fitness functions: division, movement and vibration.…”

Section: Using 'In Silico' Algorithms To Program Dropletsmentioning

confidence: 99%

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Towards heterotic computing with droplets in a fully automated droplet-maker platform

Henson

Gutiérrez

Hinkley

et al. 2015

Phil. Trans. R. Soc. A.

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The control and prediction of complex chemical systems is a difficult problem due to the nature of the interactions, transformations and processes occurring. From self-assembly to catalysis and self-organization, complex chemical systems are often heterogeneous mixtures that at the most extreme exhibit system-level functions, such as those that could be observed in a living cell. In this paper, we outline an approach to understand and explore complex chemical systems using an automated droplet maker to control the composition, size and position of the droplets in a predefined chemical environment. By investigating the spatio-temporal dynamics of the droplets, the aim is to understand how to control system-level emergence of complex chemical behaviour and even view the system-level behaviour as a programmable entity capable of information processing. Herein, we explore how our automated droplet-maker platform could be viewed as a prototype chemical heterotic computer with some initial data and example problems that may be viewed as potential chemically embodied computations.

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