Short Synthetic Terminators for Assembly of Transcription Units in Vitro and Stable Chromosomal Integration in Yeast S. cerevisiae.

Assembly of synthetic genetic circuits is central to synthetic biology. Yeast S. cerevisiae, in particular, has proven to be an ideal chassis for synthetic genome assemblies by exploiting its efficient homologous recombination. However, this property of efficient homologous recombination poses a problem for multigene assemblies in yeast, since repeated usage of standard parts, such as transcriptional terminators, can lead to rearrangements of the repeats in assembled DNA constructs in vivo. To address this issue in developing a library of orthogonal genetic components for yeast, we designed a set of short synthetic terminators based on a consensus sequence with random linkers to avoid repetitive sequences. We constructed a series of expression vectors with these synthetic terminators for efficient assembly of synthetic genes using Gateway recombination reactions. We also constructed two BAC (bacterial artificial chromosome) vectors for assembling multiple transcription units with the synthetic terminators in vitro and their integration in the yeast genome. The tandem array of synthetic genes integrated in the genome by this method is highly stable because there are few homologous segments in the synthetic constructs. Using this system of assembly and genomic integration of transcription units, we tested the synthetic terminators and their influence on the proximal transcription units. Although all the synthetic terminators have the common consensus with the identical length, they showed different activities and impacts on the neighboring transcription units.

Synthetic biology routes to bio-artificial intelligence.

The design of synthetic gene networks (SGNs) has advanced to the extent that novel genetic circuits are now being tested for their ability to recapitulate archetypal learning behaviours first defined in the fields of machine and animal learning. Here, we discuss the biological implementation of a perceptron algorithm for linear classification of input data. An expansion of this biological design that encompasses cellular 'teachers' and 'students' is also examined. We also discuss implementation of Pavlovian associative learning using SGNs and present an example of such a scheme and in silico simulation of its performance. In addition to designed SGNs, we also consider the option to establish conditions in which a population of SGNs can evolve diversity in order to better contend with complex input data. Finally, we compare recent ethical concerns in the field of artificial intelligence (AI) and the future challenges raised by bio-artificial intelligence (BI).

Accurate chromosome segregation by probabilistic self-organisation.

BACKGROUND: For faithful chromosome segregation during cell division, correct attachments must be established between sister chromosomes and microtubules from opposite spindle poles through kinetochores (chromosome bi-orientation). Incorrect attachments of kinetochore microtubules (kMTs) lead to chromosome mis-segregation and aneuploidy, which is often associated with developmental abnormalities such as Down syndrome and diseases including cancer. The interaction between kinetochores and microtubules is highly dynamic with frequent attachments and detachments. However, it remains unclear how chromosome bi-orientation is achieved with such accuracy in such a dynamic process. RESULTS: To gain new insight into this essential process, we have developed a simple mathematical model of kinetochore-microtubule interactions during cell division in general, i.e. both mitosis and meiosis. Firstly, the model reveals that the balance between attachment and detachment probabilities of kMTs is crucial for correct chromosome bi-orientation. With the right balance, incorrect attachments are resolved spontaneously into correct bi-oriented conformations while an imbalance leads to persistent errors. In addition, the model explains why errors are more commonly found in the first meiotic division (meiosis I) than in mitosis and how a faulty conformation can evade the spindle assembly checkpoint, which may lead to a chromosome loss. CONCLUSIONS: The proposed model, despite its simplicity, helps us understand one of the primary causes of chromosomal instability-aberrant kinetochore-microtubule interactions. The model reveals that chromosome bi-orientation is a probabilistic self-organisation, rather than a sophisticated process of error detection and correction.

Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae.

Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene's functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology.

Mathematical modeling of gene regulatory networks in Xenopus development.

Mathematical modeling has become increasingly indispensable for scientists who study the dynamics of gene regulatory networks (GRN) that underlie cell differentiation and pattern formation in animal development including Xenopus embryogenesis. Here I outline a step-by-step procedure for constructing a mathematical model of GRN based on ordinary differential equations (ODE), using the network of Activin and its downstream target genes Xenopus Brachyury (Xbra) and Goosecoid (Gsc) as an example. I also briefly explain methods to analyse the dynamics described by an ODE model.

Theoretical basis of the community effect in development.

BACKGROUND: Genetically identical cells often show significant variation in gene expression profile and behaviour even in the same physiological condition. Notably, embryonic cells destined to the same tissue maintain a uniform transcriptional regulatory state and form a homogeneous cell group. One mechanism to keep the homogeneity within embryonic tissues is the so-called community effect in animal development. The community effect is an interaction among a group of many nearby precursor cells, and is necessary for them to maintain tissue-specific gene expression and differentiate in a coordinated manner. Although it has been shown that the cell-cell communication by a diffusible factor plays a crucial role, it is not immediately obvious why a community effect needs many cells. RESULTS: In this work, we propose a model of the community effect in development, which consists in a linear gene cascade and cell-cell communication. We examined the properties of the model theoretically using a combination of stochastic and deterministic modelling methods. We have derived the analytical formula for the threshold size of a cell population that is necessary for a community effect, which is in good agreement with stochastic simulation results. CONCLUSIONS: Our theoretical analysis indicates that a simple model with a linear gene cascade and cell-cell communication is sufficient to reproduce the community effect in development. The model explains why a community needs many cells. It suggests that the community's long-term behaviour is independent of the initial induction level, although the initiation of a community effect requires a sufficient amount of inducing signal. The mechanism of the community effect revealed by our theoretical analysis is analogous to that of quorum sensing in bacteria. The community effect may underlie the size control in animal development and also the genesis of autosomal dominant diseases including tumorigenesis.

Visualizing protein interactions by bimolecular fluorescence complementation in Xenopus.

Bimolecular fluorescence complementation (BiFC) provides a simple and direct way to visualise protein-protein interactions in vivo and in real-time. In this article, we describe methods by which one can implement this approach in embryos of the South African claw-toed frog Xenopus laevis. We have made use of Venus, an improved version of yellow fluorescent protein (YFP), so as to achieve rapid detection of protein interactions. To suppress spontaneous interactions between the N- and C-terminal fragments of Venus, a point mutation (T153M) was introduced into the N-terminal fragment. We have used this reagent to monitor signalling by members of the transforming growth factor type beta family in cells of the Xenopus embryo.

A mechanism regulating the onset of Sox2 expression in the embryonic neural plate.

In vertebrate embryos, the earliest definitive marker for the neural plate, which will give rise to the entire central nervous system, is the transcription factor Sox2. Although some of the extracellular signals that regulate neural plate fate have been identified, we know very little about the mechanisms controlling Sox2 expression and thus neural plate identity. Here, we use electroporation for gain- and loss-of-function in the chick embryo, in combination with bimolecular fluorescence complementation, two-hybrid screens, chromatin immunoprecipitation, and reporter assays to study protein interactions that regulate expression of N2, the earliest enhancer of Sox2 to be activated and which directs expression to the largest part of the neural plate. We show that interactions between three coiled-coil domain proteins (ERNI, Geminin, and BERT), the heterochromatin proteins HP1alpha and HP1gamma acting as repressors, and the chromatin-remodeling enzyme Brm acting as activator control the N2 enhancer. We propose that this mechanism regulates the timing of Sox2 expression as part of the process of establishing neural plate identity.

Nuclear accumulation of Smad complexes occurs only after the midblastula transition in Xenopus.

Activin and the Nodal-related proteins induce mesendodermal tissues during Xenopus development. These signals act through specific receptors to cause the phosphorylation, at their carboxyl termini, of Smad2 and Smad3. The phosphorylated Smad proteins form heteromeric complexes with Smad4 and translocate into the nucleus to activate the transcription, after the midblastula transition, of target genes such as Xbra and goosecoid (gsc). In this paper we use bimolecular fluorescence complementation (BiFC) to study complex formation between Smad proteins both in vivo and in response to exogenous proteins. The technique has allowed us to detect Smad2-Smad4 heteromeric interactions during normal Xenopus development and Smad2 and Smad4 homo- and heteromers in isolated Xenopus blastomeres. Smad2-Smad2 and Smad2-Smad4 complexes accumulate rapidly in the nuclei of responding cells following Activin treatment, whereas Smad4 homomeric complexes remain cytoplasmic. When cells divide, Smad2-Smad4 complexes associate with chromatin, even in the absence of ligand. Our observation that Smad2-Smad4 complexes accumulate in the nucleus only after the midblastula transition, irrespective of the stage at which cells were treated with Activin, may shed light on the mechanisms of developmental timing.

A mechanism for the sharp transition of morphogen gradient interpretation in Xenopus.

BACKGROUND: One way in which positional information is established during embryonic development is through the graded distribution of diffusible morphogens. Unfortunately, little is known about how cells interpret different concentrations of morphogen to activate different genes or how thresholds are generated in a morphogen gradient. RESULTS: Here we show that the concentration-dependent induction of the T-box transcription factor Brachyury (Xbra) and the homeobox-containing gene Goosecoid (Gsc) by activin in Xenopus can be explained by the dynamics of a simple network consisting of three elements with a mutual negative feedback motif that can function to convert a graded signal (activin) into a binary output (Xbra on and Gsc off, or vice versa). Importantly, such a system can display sharp thresholds. Consistent with the predictions of our model, Xenopus ectodermal cells display a binary response at the single cell level after treatment with activin. CONCLUSION: This kind of simple network with mutual negative feedback might provide a general mechanism for selective gene activation in response to different levels of a single external signal. It provides a mechanism by which a sharp boundary might be created between domains of different cell types in response to a morphogen gradient.

A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system.

The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development.