Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse, and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.
A simple circuit for cost-benefit decision derived from behavioral and neural studies of the predatory sea-slug Pleurobranchaea may closely resemble that upon which the more complex valuation and decision processes of the social vertebrates are built. The neuronal natures of the pathways in the connectionist model comprise classic central pattern generators, bipolar switch mechanisms, and neuromodulatory state regulation. Marked potential exists for exploring more complex neuroeconomic behavior by appending appropriate circuitry in simulo.
A rudimentary aesthetic sense is found in the stimulus valuations and cost-benefit decisions made by primitive generalist foragers. These are based on factors governing personal economic decisions: incentive, appetite, and learning. We find that the addictive process is an extreme expression of aesthetic dynamics. An interactive, agent-based model, ASIMOV, reproduces a simple aesthetic sense from known neural relations of cost-benefit decision in foraging. In the presence of very high reward, an addiction-like process emerges. A drug-like prey provides extreme reward with no nutritive value, initiating high selectivity and prolonged cravings for drug through reward learning. Varying reward experience, caused by homeostatic changes in the neural circuitry of reward, further establishes the course of addiction, consisting of desensitization, withdrawal, resensitization, and associated changes in nutritional choice and pain sensitivity. These observations are consistent with the early evolution of addiction mechanisms in simple generalist foragers as an aesthetic sense for evaluating prey. ASIMOV is accessible to inspection, modification, and experiment, is adaptable as an educational tool, and provides insight on the possible coevolutionary origins of aesthetics and the addiction process. The aesthetic sense is a subjective, evaluative faculty used to distinguish positive and negative qualities of situations, objects, and constructs, and to bias behavioral decision toward or away from those stimuli. It is based on built-in preferences and feature detection, as well as learned preferences established from experience through reward learning. In humans, the highly developed aesthetic sense extends from judgements of taste and beauty to disgust. In other animals, it notably functions in mate choice, nest building, and foraging. Darwin and others 1,2 attributed the origin of the aesthetic sense to mate choice and reproductive displays, as are notable in many vertebrates. However, here we explore the ramifications of a potentially earlier origin in the foraging decisions of generalist animal species, where valuations of potential prey are made in estimates of nutritional value that factor in need, learned attributes, and risk. A primitive basis of the aesthetic sense appeared in our studies of the neuronal circuitry of decision in the predatory, generalist sea slug, Pleurobranchaea californica, in the animal's ability to evaluate stimuli in contexts of motivation and reward learning. It was initially implemented in an agent-based foraging simulation, Cyberslug 3. That agent made foraging decisions for approach or avoidance like the real animal, based on stimulus quality, motivation, and reward learning, and satisfied requirements of optimized foraging. We introduced the original simulation as an example of simple neuronal relations that could be elaborated for more complex cognition and behavior, as may have happened to ancestral bilaterians in evolution. Accordingly, here we introduce a new version, ASIMOV, which is upg...
Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers.Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs.Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.
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