A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging.

Fluorescent tools have revolutionized our ability to probe biological dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-molecule dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small molecules, and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell analysis and especially live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.

Differentiation and Maturation of Muscle and Fat Cells in Cultivated Seafood: Lessons from Developmental Biology.

Cultivated meat, also known as cultured or cell-based meat, is meat produced directly from cultured animal cells rather than from a whole animal. Cultivated meat and seafood have been proposed as a means of mitigating the substantial harms associated with current production methods, including damage to the environment, antibiotic resistance, food security challenges, poor animal welfare, and-in the case of seafood-overfishing and ecological damage associated with fishing and aquaculture. Because biomedical tissue engineering research, from which cultivated meat draws a great deal of inspiration, has thus far been conducted almost exclusively in mammals, cultivated seafood suffers from a lack of established protocols for producing complex tissues in vitro. At the same time, fish such as the zebrafish Danio rerio have been widely used as model organisms in developmental biology. Therefore, many of the mechanisms and signaling pathways involved in the formation of muscle, fat, and other relevant tissue are relatively well understood for this species. The same processes are understood to a lesser degree in aquatic invertebrates. This review discusses the differentiation and maturation of meat-relevant cell types in aquatic species and makes recommendations for future research aimed at recapitulating these processes to produce cultivated fish and shellfish.

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges.

Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high-quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties.

De novo design of peptide-calcite biomineralization systems.

Many organisms produce complex, hierarchically structured, inorganic materials via protein-influenced crystal growth--a process known as biomineralization. Understanding this process would shed light on hard-tissue formation and guide efforts to develop biomaterials. We created and tested a computational method to design protein-biomineralization systems. The algorithm folds a protein from a fully extended structure and simultaneously optimizes the fold, orientation, and sequence of the protein adsorbed to a crystal surface. We used the algorithm to design peptides (16 residues) to modify calcite (CaCO(3)) crystallization. We chemically synthesized six peptides that were predicted to bind different states of a calcite growth plane. All six peptides dramatically affected calcite crystal growth (as observed by scanning electron microscopy), and the effects were dependent on the targeted state of the {001} growth plane. Additionally, we synthesized and assayed scrambled variants of all six designed peptides to distinguish cases where sequence composition determines the interactions versus cases where sequence order (and presumably structure) plays a role. Scrambled variants of negatively charged peptides also had dramatic effects on calcite crystallization; in contrast, scrambled variants of positively charged peptides had a variable effect on crystallization, ranging from dramatic to mild. Special emphasis is often placed on acidic protein residues in calcified tissue mineralization; the work presented here suggests an important role for basic residues as well. In particular, this work implicates a potential role for basic residues in sequence-order specificity for peptide-mineral interactions.

An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas.

The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains.

Algae-based oral recombinant vaccines.

Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for "molecular pharming" in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae could be poised to become the next candidate in recombinant subunit vaccine production, as they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered - from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and systemic immune reactivity.

Synthetic oligonucleotide libraries reveal novel regulatory elements in Chlamydomonas chloroplast mRNAs.

Gene expression in chloroplasts is highly regulated during translation by sequence and secondary-structure elements in the 5' untranslated region (UTR) of mRNAs. These chloroplast mRNA 5' UTRs interact with nuclear-encoded factors to regulate mRNA processing, stability, and translation initiation. Although several UTR elements in chloroplast mRNAs have been identified by site-directed mutagenesis, the complete set of elements required for expression of plastid mRNAs remains undefined. Here we present a synthetic biology approach using an arrayed oligonucleotide library to examine in vivo hundreds of designed variants of endogenous UTRs from Chlamydomonas reinhardtii and quantitatively identify essential regions through next-generation sequencing of thousands of mutants. We validate this strategy by characterizing the relatively well-studied 5' UTR of the psbD mRNA encoding the D2 protein in photosystem II and find that our analysis generally agrees with previous work identifying regions of importance but significantly expands and clarifies the boundaries of these regulatory regions. We then use this strategy to characterize the previously unstudied psaA 5' UTR and obtain a detailed map of regions essential for both positive and negative regulation. This analysis can be performed in a high-throughput manner relative to previous site-directed mutagenesis methods, enabling compilation of a large unbiased data set of regulatory elements of chloroplast gene expression. Finally, we create a novel synthetic UTR based on aggregate sequence analysis from the libraries and demonstrate that it significantly increases accumulation of an exogenous protein, attesting to the utility of this strategy for enhancing protein production in algal chloroplasts.

Advances in microalgae engineering and synthetic biology applications for biofuel production.

Among the technologies being examined to produce renewable fuels, microalgae are viewed by many in the scientific community as having the greatest potential to become economically viable. Algae are capable of producing greater than 50,000 kg/acre/year of biomass [1]. Additionally, most algae naturally accumulate energy-dense oils that can easily be converted into transportation fuels. To reach economic parity with fossil fuels there are still several challenges. These include identifying crop protection strategies, improving harvesting and oil extraction processes, and increasing biomass productivity and oil content. All of these challenges can be impacted by genetic, molecular, and ultimately synthetic biology techniques, and all of these technologies are being deployed to enable algal biofuels to become economically competitive with fossil fuels.