Preparation of Chromobodies for the Detection of Cell Surface Epitopes.

Chromobodies are nanobodies genetically fused to fluorescent proteins, which were developed to visualize endogenous intracellular antigens. These versatile bioimaging nanotools can also be used to detect cell surface epitopes, and we describe here how we use them as an alternative to conjugated antibodies. This way, we routinely test the binding efficiency of nanobodies for their cognate cell surface antigens, before integrating them as sensing domains into complex synthetic receptor architectures.

Improving recombinant protein production in CHO cells using the CRISPR-Cas system.

Chinese hamster ovary (CHO) cells are among the most widely used mammalian cell lines in the biopharmaceutical industry. Therefore, it is not surprising that significant efforts have been made around the engineering of CHO cells using genetic engineering methods such as the CRISPR-Cas system. In this review, we summarize key recent studies that have used different CRISPR-Cas systems such as Cas9, Cas13 or dCas9 fused with effector domains to improve recombinant protein (r-protein) production in CHO cells. Here, every relevant stage of production was considered, underscoring the advantages and limitations of these systems, as well as discussing their bottlenecks and probable solutions. A special emphasis was given on how these systems could disrupt and/or regulate genes related to glycan composition, which has relevant effects over r-protein properties and in vivo activity. Furthermore, the related promising future applications of CRISPR to achieve a tunable, reversible, or highly stable editing of CHO cells are discussed. Overall, the studies covered in this review show that despite the complexity of mammalian cells, the synthetic biology community has developed many mature strategies to improve r-protein production using CHO cells. In this regard, CRISPR-Cas technology clearly provides efficient and flexible genetic manipulation and allows for the generation of more productive CHO cell lines, leading to more cost-efficient production of biopharmaceuticals, however, there is still a need for many emerging techniques in CRISPR to be reported in CHO cells; therefore, more research in these cells is needed to realize the full potential of this technology.

Bioengineering Self-Organizing Signaling Centers to Control Embryoid Body Pattern Elaboration.

Multicellular systems possess an intrinsic capacity to autonomously generate nonrandom state distributions or morphologies in a process termed self-organization. Facets of self-organization, such as pattern formation, pattern elaboration, and symmetry breaking, are frequently observed in developing embryos. Artificial stem cell-derived structures including embryoid bodies (EBs), gastruloids, and organoids also demonstrate self-organization, but with a limited capacity compared to their in vivo developmental counterparts. There is a pressing need for better tools to allow user-defined control over self-organization in these stem cell-derived structures. Here, we employ synthetic biology to establish an efficient platform for the generation of self-organizing coaggregates, in which HEK-293 cells overexpressing P-cadherin (Cdh3) spontaneously form cell clusters attached mostly to one or two locations on the exterior of EBs. These Cdh3-expressing HEK cells, when further engineered to produce functional mouse WNT3A, evoke polarized and gradual Wnt/ß-catenin pathway activation in EBs during coaggregation cultures. The localized WNT3A provision induces nascent mesoderm specification within regions of the EB close to the Cdh3-Wnt3a-expressing HEK source, resulting in pattern elaboration and symmetry breaking within EBs. This synthetic biology-based approach puts us closer toward engineering synthetic organizers to improve the realism in stem cell-derived structures.

Crossing Kingdoms: How Can Art Open Up New Ways of Thinking About Science?

"Crossing Kingdoms" is an artist-led experiment in the biological fusion of mammalian and yeast cells and the cultural discussions of these phenomena. We present this collaboration as an experiment in responsible research and innovation (RRI), an institutionalized format for ensuring that researchers reflect on the wider social dimensions of their work. Our methods challenged us as researchers to reflect on interdisciplinary collaboration and the possibility of innovating in biology for artistic purposes, challenged audiences to reflect on biological boundaries, and challenged both groups to reflect on what it means to be responsible in science. We conclude that our experiment in RRI was successful because we have asked unexpected questions-a contrast to RRI implemented as a standard protocol. Our experiment has implications for biologists and artists pursuing interdisciplinary collaborations with each other and for researchers thinking about implementing RRI as more than a box-ticking exercise.

Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells.

Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch ('Blue-OFF'), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.

Synthetic biology meets tissue engineering.

Classical tissue engineering is aimed mainly at producing anatomically and physiologically realistic replacements for normal human tissues. It is done either by encouraging cellular colonization of manufactured matrices or cellular recolonization of decellularized natural extracellular matrices from donor organs, or by allowing cells to self-organize into organs as they do during fetal life. For repair of normal bodies, this will be adequate but there are reasons for making unusual, non-evolved tissues (repair of unusual bodies, interface to electromechanical prostheses, incorporating living cells into life-support machines). Synthetic biology is aimed mainly at engineering cells so that they can perform custom functions: applying synthetic biological approaches to tissue engineering may be one way of engineering custom structures. In this article, we outline the 'embryological cycle' of patterning, differentiation and morphogenesis and review progress that has been made in constructing synthetic biological systems to reproduce these processes in new ways. The state-of-the-art remains a long way from making truly synthetic tissues, but there are now at least foundations for future work.

2- and 3-dimensional synthetic large-scale de novo patterning by mammalian cells through phase separation.

Synthetic biology provides an opportunity for the construction and exploration of alternative solutions to biological problems - solutions different from those chosen by natural life. To this end, synthetic biologists have built new sensory systems, cellular memories, and alternative genetic codes. There is a growing interest in applying synthetic approaches to multicellular systems, especially in relation to multicellular self-organization. Here we describe a synthetic biological system that confers large-scale de novo patterning activity on 2-D and 3-D populations of mammalian cells. Instead of using the reaction-diffusion mechanisms common in real embryos, our system uses cadherin-mediated phase separation, inspired by the known phenomenon of cadherin-based sorting. An engineered self-organizing, large-scale patterning system requiring no prior spatial cue may be a significant step towards the construction of self-assembling synthetic tissues.

A library of mammalian effector modules for synthetic morphology.

BACKGROUND: In mammalian development, the formation of most tissues is achieved by a relatively small repertoire of basic morphogenetic events (e.g. cell adhesion, locomotion, apoptosis, etc.), permutated in various sequences to form different tissues. Together with cell differentiation, these mechanisms allow populations of cells to organize themselves into defined geometries and structures, as simple embryos develop into complex organisms. The control of tissue morphogenesis by populations of engineered cells is a potentially very powerful but neglected aspect of synthetic biology. RESULTS: We have assembled a modular library of synthetic morphogenetic driver genes to control (separately) mammalian cell adhesion, locomotion, fusion, proliferation and elective cell death. Here we describe this library and demonstrate its use in the T-REx-293 human cell line to induce each of these desired morphological behaviours on command. CONCLUSIONS: Building on from the simple test systems described here, we want to extend engineered control of morphogenetic cell behaviour to more complex 3D structures that can inform embryologists and may, in the future, be used in surgery and regenerative medicine, making synthetic morphology a powerful tool for developmental biology and tissue engineering.

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis.

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.

Lactobacillus suntoryeus sp. nov., isolated from malt whisky distilleries.

Eight strains of Lactobacillus with identical partial 16S rRNA gene sequences and similar randomly amplified polymorphic DNA patterns were isolated from fermentation samples from Japanese and Scottish malt whisky distilleries. Phylogenetic analysis of almost complete 16S rRNA gene sequences from three representative strains (two from Japan, one from Scotland) placed them in the genus Lactobacillus as members of the Lactobacillus acidophilus group. Lactobacillus helveticus and Lactobacillus gallinarum were the most closely related species, with 16S rRNA gene similarities of 99.3 and 98.1 %, respectively. A similar phylogeny was derived from partial sequences of elongation factor Tu (tuf) genes in which the alleles from the three distillery isolates were identical and shared 99.0 % similarity with L. helveticus and L. gallinarum tuf genes. S-layer (slp) gene sequences suggested different relationships among the strains and the distillery isolates no longer formed a monophyletic group. The alleles from the Japanese and Scottish strains shared only 54 % similarity. Chromosomal DNA from the distillery strains gave DNA-DNA hybridization values between 79 and 100 % but showed less than 43 and 22 % reassociation with L. helveticus and L. gallinarum DNA, respectively. The name Lactobacillus suntoryeus sp. nov. is proposed for this novel taxon; the type strain is strain SAT (=LMG 22464T=NCIMB 14005T).