Implementing re-configurable biological computation with distributed multicellular consortia.

The use of synthetic biological circuits to deal with numerous biological challenges has been proposed in several studies, but its implementation is still remote. A major problem encountered is the complexity of the cellular engineering needed to achieve complex biological circuits and the lack of general-purpose biological systems. The generation of re-programmable circuits can increase circuit flexibility and the scalability of complex cell-based computing devices. Here we present a new architecture to produce reprogrammable biological circuits that allow the development of a variety of different functions with minimal cell engineering. We demonstrate the feasibility of creating several circuits using only a small set of engineered cells, which can be externally reprogrammed to implement simple logics in response to specific inputs. In this regard, depending on the computation needs, a device composed of a number of defined cells can generate a variety of circuits without the need of further cell engineering or rearrangements. In addition, the inclusion of a memory module in the circuits strongly improved the digital response of the devices. The reprogrammability of biological circuits is an intrinsic capacity that is not provided in electronics and it may be used as a tool to solve complex biological problems.

Implementation of Complex Biological Logic Circuits Using Spatially Distributed Multicellular Consortia.

Engineered synthetic biological devices have been designed to perform a variety of functions from sensing molecules and bioremediation to energy production and biomedicine. Notwithstanding, a major limitation of in vivo circuit implementation is the constraint associated to the use of standard methodologies for circuit design. Thus, future success of these devices depends on obtaining circuits with scalable complexity and reusable parts. Here we show how to build complex computational devices using multicellular consortia and space as key computational elements. This spatial modular design grants scalability since its general architecture is independent of the circuit's complexity, minimizes wiring requirements and allows component reusability with minimal genetic engineering. The potential use of this approach is demonstrated by implementation of complex logical functions with up to six inputs, thus demonstrating the scalability and flexibility of this method. The potential implications of our results are outlined.

Distributed biological computation with multicellular engineered networks.

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.

A bottom-up characterization of transfer functions for synthetic biology designs: lessons from enzymology.

Within the field of synthetic biology, a rational design of genetic parts should include a causal understanding of their input-output responses-the so-called transfer function-and how to tune them. However, a commonly adopted strategy is to fit data to Hill-shaped curves without considering the underlying molecular mechanisms. Here we provide a novel mathematical formalization that allows prediction of the global behavior of a synthetic device by considering the actual information from the involved biological parts. This is achieved by adopting an enzymology-like framework, where transfer functions are described in terms of their input affinity constant and maximal response. As a proof of concept, we characterize a set of Lux homoserine-lactone-inducible genetic devices with different levels of Lux receptor and signal molecule. Our model fits the experimental results and predicts the impact of the receptor's ribosome-binding site strength, as a tunable parameter that affects gene expression. The evolutionary implications are outlined.

Lack of Bmal1 leads to changes in rhythmicity and impairs motivation towards natural stimuli.

Maintaining proper circadian rhythms is essential for coordinating biological functions in mammals. This study investigates the effects of daily arrhythmicity using Bmal1-knockout (KO) mice as a model, aiming to understand behavioural and motivational implications. By employing a new mathematical analysis based on entropy divergence, we identified disrupted intricate activity patterns in mice derived by the complete absence of BMAL1 and quantified the difference regarding the activity oscillation's complexity. Changes in locomotor activity coincided with disturbances in circadian gene expression patterns. Additionally, we found a dysregulated gene expression profile particularly in brain nuclei like the ventral striatum, impacting genes related to reward and motivation. Further investigation revealed that arrhythmic mice exhibited heightened motivation for food and water rewards, indicating a link between circadian disruptions and the reward system. This research sheds light on how circadian clock alterations impact the gene expression regulating the reward system and how this, in turn, can lead to altered seeking behaviour and motivation for natural rewards. In summary, the present study contributes to our understanding of how reward processing is under the regulation of circadian clock machinery.

Evolutionary algorithm for the optimization of meal intake and insulin administration in patients with type 2 diabetes.

The optimal management of type 2 diabetes (T2DM) is complex and involves an appropriate combination of diet, exercise, and different pharmacological treatments. Artificial intelligence-based tools have been shown to be very useful for the diagnosis and treatment of diverse pathologies, including diabetes. In the present study, we present a proof of concept of the potential of an evolutionary algorithm to optimize the meal size, timing and insulin dose for the control of glycemia. We found that an appropriate distribution of food intake throughout the day permits a reduction in the insulin dose required to maintain glycemia within the range recommended by the American Diabetes Association for patients with T2DM of a range of severities. Furthermore, the effects of restrictions to both the timing and amount of food ingested were assessed, and we found that an increase in the amount of insulin was required to control glycemia as dietary intake became more restricted. In the near future, the use of these computational tools should permit patients with T2DM to optimize their personal meal schedule and insulin dose, according to the severity of their diabetes.

Development and evaluation of culture media based on extracts of the cyanobacterium Arthrospira platensis.

Continuous advances in the fields of industrial biotechnology and pharmacy require the development of new formulations of culture media based on new nutrient sources. These new sources must be sustainable, high yielding, and non-animal-based, with minimal environmental impact. Thus, culture media prepared from cyanobacterial extracts can be an interesting alternative to the current formulations. In this study, we prepared various minimal formulations of culture media using the extracts of Arthrospira platensis, and analyzed the efficiency of these formulations, based on their effect on the production of biomass and molecules of industrial interest, using different types of bacteria. All media formulations prepared in this study showed better performance than conventional media, including those based on animal ingredients. Thus, based on their versatility and high-yielding capacity, we conclude that culture media prepared from cyanobacterial extracts are a good alternative to conventional media for meeting the current demands of the cosmetic and pharmaceutical industries.

2D printed multicellular devices performing digital and analogue computation.

Much effort has been expended on building cellular computational devices for different applications. Despite the significant advances, there are still several addressable restraints to achieve the necessary technological transference. These improvements will ease the development of end-user applications working out of the lab. In this study, we propose a methodology for the construction of printable cellular devices, digital or analogue, for different purposes. These printable devices are designed to work in a 2D surface, in which the circuit information is encoded in the concentration of a biological signal, the so-called carrying signal. This signal diffuses through the 2D surface and thereby interacts with different device components. These components are distributed in a specific spatial arrangement and perform the computation by modulating the level of the carrying signal in response to external inputs, determining the final output. For experimental validation, 2D cellular circuits are printed on a paper surface by using a set of cellular inks. As a proof-of-principle, we have printed and analysed both digital and analogue circuits using the same set of cellular inks but with different spatial topologies. The proposed methodology can open the door to a feasible and reliable industrial production of cellular circuits for multiple applications.

General Analyses of Gene Expression Dependencies on Genetic Burden.

Over the last decade, the combining of newly developed molecular tools for DNA editing with engineering principles has allowed the creation of complex cellular devices, usually based on complex genetic circuits, for many different purposes. However, when the technological evolution of genetic circuitry is compared with previous technologies such as electronic circuitry, clear limitations regarding the technological scalability of genetic circuitry are observed due to the lack of predictability. To overcome this problem, it is necessary to create new theoretical frameworks for designing genetic circuits in a feasible and reliable manner, taking into account those limitations. Among a number of such limitations, the so-called genetic burden is one of the main constraints. Surprisingly, despite its relevance, little attention has been paid to genetic burden, and it is often not considered when designing genetic circuits. In this study, a new general mathematical formalism is presented, describing the effects of genetic burden on gene expression. The mathematical analysis shows that alterations in gene expression due to genetic burden can be qualitatively described independently of the specific genetic features of the system under consideration. The mathematical model was experimentally tested in different genetic circuits. The experimental evidence coincides with the expected behaviors described by the model in complex scenarios. For instance, observed modulations in the expression levels of constitutive genes in response to changes in the levels of external inducers of gene expression that do not directly modulate them, or the emergence of limitations in gene overexpression, can be understood in terms of genetic burden. The present mathematical formalism provides a useful general framework for gene circuit design that will help to advance synthetic biological systems.

Two-Component Biosensors: Unveiling the Mechanisms of Predictable Tunability.

Many studies have been devoted to the engineering of cellular biosensors by exploiting intrinsic natural sensors. However, biosensors rely not only on input detection but also on an adequate response range. It is therefore often necessary to tune natural systems to meet the demands of specific applications in a predictable manner. In this study, we explored the customizability of two-component bacterial biosensors by modulating the main biosensor component, i.e., the receptor protein. We developed a mathematical model that describes the functional relationship between receptor abundance and activation threshold, sensitivity, dynamic range, and operating range. The defined mathematical framework allows the design of the genetic architecture of a two-component biosensor that can perform as required with minimal genetic engineering. To experimentally validate the model and its predictions, a library of biosensors was constructed. The good agreement between theoretical designs and experimental results indicates that modulation of receptor protein abundance allows optimization of biosensor designs with minimal genetic engineering.

Why are cellular switches Boolean? General conditions for multistable genetic circuits.

The logic of cellular decision-making is largely controlled by regulatory circuits defining molecular switches. Such switching elements allow to turn a graded input signal into an all-or-nothing output. Traditional studies have focused on this bistable picture of regulation, but higher-order scenarios involving tristable and tetrastable states are possible too. Are these multiswitches allowed in simple gene regulatory networks? Are they likely to be observed? If not, why not? In this paper we present the examination of this question by means of a simple but powerful geometric approach. We examine the relation between multistability, the degree of multimerization of the regulators and the role of autoloops within a deterministic setting, finding that N-stable circuits are possible, although their likelihood to occur rapidly decays with the order of the switch. Our work indicates that, despite two-component circuits are able to implement multistability, they are optimal for Boolean switches. The evolutionary implications are outlined.

Distributed robustness in cellular networks: insights from synthetic evolved circuits.

Evolved natural systems are known to display some sort of distributed robustness against the loss of individual components. Such type of robustness is not just the result of redundancy. Instead, it seems to be based on degeneracy, i.e. the ability of elements that are structurally different to perform the same function or yield the same output. Here, we explore the problem of how relevant is degeneracy in a class of evolved digital systems formed by NAND gates, and what types of network structures underlie the resilience of evolved designs to the removal or loss of a given unit. It is shown that our fault tolerant circuits are obtained only if robustness arises in a distributed manner. No such reliable systems were reached just by means of redundancy, thus suggesting that reliable designs are necessarily tied to degeneracy.

Plug-and-Play Multicellular Circuits with Time-Dependent Dynamic Responses.

Synthetic biology studies aim to develop cellular devices for biomedical applications. These devices, based on living instead of electronic or electromechanic technology, might provide alternative treatments for a wide range of diseases. However, the feasibility of these devices depends, in many cases, on complex genetic circuits that must fulfill physiological requirements. In this work, we explored the potential of multicellular architectures to act as an alternative to complex circuits for implementation of new devices. As a proof of concept, we developed specific circuits for insulin or glucagon production in response to different glucose levels. Here, we show that fundamental features, such as circuit's affinity or sensitivity, are dependent on the specific configuration of the multicellular consortia, providing a method for tuning these properties without genetic engineering. As an example, we have designed and built circuits with an incoherent feed-forward loop architecture (FFL) that can be easily adjusted to generate single pulse responses. Our results might serve as a blueprint for future development of cellular devices for glycemia regulation in diabetic patients.

Synthetic associative learning in engineered multicellular consortia.

Associative learning (AL) is one of the key mechanisms displayed by living organisms in order to adapt to their changing environments. It was recognized early as a general trait of complex multicellular organisms but is also found in 'simpler' ones. It has also been explored within synthetic biology using molecular circuits that are directly inspired in neural network models of conditioning. These designs involve complex wiring diagrams to be implemented within one single cell, and the presence of diverse molecular wires become a challenge that might be very difficult to overcome. Here we present three alternative circuit designs based on two-cell microbial consortia able to properly display AL responses to two classes of stimuli and displaying long- and short-term memory (i.e. the association can be lost with time). These designs might be a helpful approach for engineering the human gut microbiome or even synthetic organoids, defining a new class of decision-making biological circuits capable of memory and adaptation to changing conditions. The potential implications and extensions are outlined.

A Synthetic Multicellular Memory Device.

Changing environments pose a challenge to living organisms. Cells need to gather and process incoming information, adapting to changes in predictable ways. This requires in particular the presence of memory, which allows different internal states to be stored. Biological memory can be stored by switches that retain information on past and present events. Synthetic biologists have implemented a number of memory devices for biological applications, mostly in single cells. It has been shown that the use of multicellular consortia provides interesting advantages to implement biological circuits. Here we show how to build a synthetic biological memory switch using an eukaryotic consortium. We engineered yeast cells that can communicate and retain memory of changes in the extracellular environment. These cells were able to produce and secrete a pheromone and sense a different pheromone following NOT logic. When the two strains were cocultured, they behaved as a double-negative-feedback motif with memory. In addition, we showed that memory can be effectively changed by the use of external inputs. Further optimization of these modules and addition of other cells could lead to new multicellular circuits that exhibit memory over a broad range of biological inputs.

Computational implementation of a tunable multicellular memory circuit for engineered eukaryotic consortia.

Cells are complex machines capable of processing information by means of an entangled network of molecular interactions. A crucial component of these decision-making systems is the presence of memory and this is also a specially relevant target of engineered synthetic systems. A classic example of memory devices is a 1-bit memory element known as the flip-flop. Such system can be in principle designed using a single-cell implementation, but a direct mapping between standard circuit design and a living circuit can be cumbersome. Here we present a novel computational implementation of a 1-bit memory device using a reliable multicellular design able to behave as a set-reset flip-flop that could be implemented in yeast cells. The dynamics of the proposed synthetic circuit is investigated with a mathematical model using biologically-meaningful parameters. The circuit is shown to behave as a flip-flop in a wide range of parameter values. The repression strength for the NOT logics is shown to be crucial to obtain a good flip-flop signal. Our model also shows that the circuit can be externally tuned to achieve different memory states and dynamics, such as persistent and transient memory. We have characterized the parameter domains for robust memory storage and retrieval as well as the corresponding time response dynamics.

How to make a synthetic multicellular computer.

Biological systems perform computations at multiple scales and they do so in a robust way. Engineering metaphors have often been used in order to provide a rationale for modeling cellular and molecular computing networks and as the basis for their synthetic design. However, a major constraint in this mapping between electronic and wet computational circuits is the wiring problem. Although wires are identical within electronic devices, they must be different when using synthetic biology designs. Moreover, in most cases the designed molecular systems cannot be reused for other functions. A new approximation allows us to simplify the problem by using synthetic cellular consortia where the output of the computation is distributed over multiple engineered cells. By evolving circuits in silico, we can obtain the minimal sets of Boolean units required to solve the given problem at the lowest cost using cellular consortia. Our analysis reveals that the basic set of logic units is typically non-standard. Among the most common units, the so called inverted IMPLIES (N-Implies) appears to be one of the most important elements along with the NOT and AND functions. Although NOR and NAND gates are widely used in electronics, evolved circuits based on combinations of these gates are rare, thus suggesting that the strategy of combining the same basic logic gates might be inappropriate in order to easily implement synthetic computational constructs. The implications for future synthetic designs, the general view of synthetic biology as a standard engineering domain, as well as potencial drawbacks are outlined.

Evolvability of feed-forward loop architecture biases its abundance in transcription networks.

BACKGROUND: Transcription networks define the core of the regulatory machinery of cellular life and are largely responsible for information processing and decision making. At the small scale, interaction motifs have been characterized based on their abundance and some seemingly general patterns have been described. In particular, the abundance of different feed-forward loop motifs in gene regulatory networks displays systematic biases towards some particular topologies, which are much more common than others. The causative process of this pattern is still matter of debate. RESULTS: We analyzed the entire motif-function landscape of the feed-forward loop using the formalism developed in a previous work. We evaluated the probabilities to implement possible functions for each motif and found that the kurtosis of these distributions correlate well with the natural abundance pattern. Kurtosis is a standard measure for the peakedness of probability distributions. Furthermore, we examined the functional robustness of the motifs facing mutational pressure in silico and observed that the abundance pattern is biased by the degree of their evolvability. CONCLUSIONS: The natural abundance pattern of the feed-forward loop can be reconstructed concerning its intrinsic plasticity. Intrinsic plasticity is associated to each motif in terms of its capacity of implementing a repertoire of possible functions and it is directly linked to the motif's evolvability. Since evolvability is defined as the potential phenotypic variation of the motif upon mutation, the link plausibly explains the abundance pattern.

Distributed computation: the new wave of synthetic biology devices.

Synthetic biology (SB) offers a unique opportunity for designing complex molecular circuits able to perform predefined functions. But the goal of achieving a flexible toolbox of reusable molecular components has been shown to be limited due to circuit unpredictability, incompatible parts or random fluctuations. Many of these problems arise from the challenges posed by engineering the molecular circuitry: multiple wires are usually difficult to implement reliably within one cell and the resulting systems cannot be reused in other modules. These problems are solved by means of a nonstandard approach to single cell devices, using cell consortia and allowing the output signal to be distributed among different cell types, which can be combined in multiple, reusable and scalable ways.