Recent advances and opportunities in synthetic logic gates engineering in living cells

Recently, a number of synthetic biologic gates including AND, OR, NOR, NOT, XOR and NAND have been engineered and characterized in a wide range of hosts. The hope in the emerging synthetic biology community is to construct an inventory of well-characterized parts and install distinct gene and circuit behaviours that are externally controllable. Though the field is still growing and major successes are yet to emerge, the payoffs are predicted to be significant. In this review, we highlight specific examples of logic gates engineering with applications towards fundamental understanding of network complexity and generating a novel socially useful applications.     

Epistasis in a quantitative trait captured by a molecular model of transcription factor interactions

With technological advances in genetic mapping studies more of the genes and polymorphisms that underlie Quantitative Trait Loci (QTL) are now being identified. As the identities of these genes become known there is a growing need for an analysis framework that incorporates the molecular interactions affected by natural polymorphisms. As a step towards such a framework we present a molecular model of genetic variation in sporulation efficiency between natural isolates of the yeast, Saccharomyces cerevisiae. The model is based on the structure of the regulatory pathway that controls sporulation. The model captures the phenotypic variation between strains carrying different combinations of alleles at known QTL. Compared to a standard linear model the molecular model requires fewer free parameters, and has the advantage of generating quantitative hypotheses about the affinity of specific molecular interactions in different genetic backgrounds. Our analyses provide a concrete example of how the thermodynamic properties of protein-protein and protein-DNA interactions naturally give rise to epistasis, the non-linear relationship between genotype and phenotype. As more causative genes and polymorphisms underlying QTL are identified, thermodynamic analyses of quantitative traits may provide a useful framework for unraveling the complex relationship between genotype and phenotype.     

A simple way for sequential assignment in isotopically enriched proteins using a H(N)CACO correlation

Summary A simplified scheme for sequential assignment in isotopically enriched proteins is presented. It is based on the standard triple resonance experiments HNCO, HN(CO)CA, HNCA and a modified H(N)CACO correlation, in which both of the H^N connectivities to the CA/C pair of residue i and i-1 are observed. The H(N)CACO was tested on uniformly ¹³C/¹⁵N enriched P13 domain of mannose permease (31 kDa).     

Question 7: Construction of a Semi-Synthetic Minimal Cell: A Model for Early Living Cells

Using a Synthetic Biology approach we are building a semi-synthetic minimal cell. This represents an exercise to shape a minimal-cell model system recalling the simplicity of early living cells in early evolution. We have recently introduced into liposome compartments a minimal set of enzymes named “Puresystem” (PS) synthesizing EGFP proteins. To establish reproduction of the shell compartment with a minimal set of genes we have cloned the genes for the Fatty Acid Synthase (FAS) type I enzymes. These FAS genes introduced into liposomes, translated into FAS enzymes by PS and in the presence of precursors produce fatty acids. The resulting release of fatty acid molecules within liposome vesicles should promote vesicle growth and reproduction. The core reproduction of a minimal cell corresponding to the replication of the minimal genome will require a few genes for the DNA replication and the PS, and a minimum set of genes for the synthesis of t-RNAs. In future the reconstruction of a minimal ribosome will bring the number of genes for ribosomal proteins from 54 of an existing minimal genome down to 30–20 genes. A Synthetic Biology approach could bring the number of essential genes for a minimal cell down to 100 or less. International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Lipophosphonate/lipophosphoramidates: a family of synthetic vectors efficient for gene delivery

Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up).     

simBio: a Java package for the development of detailed cell models

Quantitative dynamic computer models, which integrate a variety of molecular functions into a cell model, provide a powerful tool to create and test working hypotheses. We have developed a new modeling tool, the simBio package (freely available from http://www.sim-bio.org/), which can be used for constructing cell models, such as cardiac cells (the Kyoto model from Matsuoka et al., 2003, 2004a, b, the LRd model from Faber and Rudy, 2000, and the Noble 98 model from Noble et al., 1998), epithelial cells (Strieter et al., 1990) and pancreatic β cells (Magnus and Keizer, 1998). The simBio package is written in Java, uses XML and can solve ordinary differential equations. In an attempt to mimic biological functional structures, a cell model is, in simBio, composed of independent functional modules called Reactors, such as ion channels and the sarcoplasmic reticulum, and dynamic variables called Nodes, such as ion concentrations. The interactions between Reactors and Nodes are described by the graph theory and the resulting graph represents a blueprint of an intricate cellular system. Reactors are prepared in a hierarchical order, in analogy to the biological classification. Each Reactor can be composed or improved independently, and can easily be reused for different models. This way of building models, through the combination of various modules, is enabled through the use of object-oriented programming concepts. Thus, simBio is a straightforward system for the creation of a variety of cell models on a common database of functional modules.     

An alternative strategy for maintenance of eusociality after nest destruction: new nest construction in a primitively eusocial wasp

Although nests are central to colonial life in social insects, nests are sometimes damaged by predators or natural disasters. After nest destruction, individuals usually construct new nests. In this case, a sophisticated mechanism like the scent trail pheromone used in large insect colonies that recruit individuals to new nest sites would be important for the maintenance of eusociality. In independent-founding Polistes wasps, it is well known that queens enforce workers physiologically on the natal nests even if evidence of trail pheromone use has not been exhibited. We investigated the effect of the queen on an alternative strategy for the maintenance of eusociality by first females after nest destruction in the primitively eusocial wasp Polistes chinensis. We predicted that the first females in queen-absent colonies have various behavioral options after nest destruction. Even if the females construct new nests cooperatively with other individuals, the new nest construction should be conducted more smoothly in queen-present colonies because the queens regulate the behavior of wasps. We made wasps construct new nests by removing the entire brood from existing nests. The presence of the queen did not cause variation in the alternative strategy of the first females, as the first females (workers) usually constructed new nests cooperatively irrespective of the queen-presence. Thus, the workers in the queenpresent colonies affiliated to the new nest construction more smoothly and constructed new nests more efficiently than workers in the queen-absent colonies. Our results suggest that the presence of the queen is important for maintaining eusociality in primitively eusocial wasps after nest destruction. Received 8 February 2005; revised 5 October 2005; accepted 17 October 2005.     

用于细胞水平家蚕转录调控元件研究的简单基础启动子的构建

【目的】本研究致力于构建一种能够在家蚕Bombyx mori细胞水平稳定表达的简单基础启动子,从而更准确地反映单一转录调控元件对基因启动子活性的影响,为研究家蚕乃至其他昆虫的基因转录调控奠定基础。【方法】本研究在本课题组已报道的能在家蚕细胞中稳定表达且基本不含上游转录调控元件的BmVgP78M启动子的基础上,通过PCR技术在其上游添加一定长度的间隔序列和能够应答20-羟基蜕皮激素(20E)且增强启动子活性的BrC-Z2转录因子结合基序(BrC-Z2 element, BrC-Z2E);通过基因克隆技术构建细胞转染载体;通过细胞转染技术和双荧光素酶报告基因系统检测启动子活性的变化。【结果】通过在BmVgP78M启动子上游添加28 bp间隔序列,成功构建了一个简单基础启动子,命名为VgP78ML,并证明其为可用于研究目标转录调控元件的简单基础启动子。经实验验证表明,该简单基础启动子不仅可以在家蚕细胞中稳定表达,且其本身活性不受20E及转录因子BrC-Z2的影响;当该启动子上游连接BrC-Z2E时,可以显著地应答20E及BrC-Z2转录因子,从而调控报告基因的表达。【结论】VgP78ML能够作为简单基础启动子应用于细胞水平对家蚕基因转录调控进行研究。同时,其构建方法也为其他物种构建研究转录调控的简单基础启动子提供了参考。     

Dissection of a major QTL for photoperiod sensitivity in rice: its association with a gene expressed in an age-dependent manner

 A proposed major quantitative trait locus (QTL) for photoperiod sensitivity on chromosome 6 in rice was examined by introducing a chromosomal segment from a sensitive line into an insensitive one. The crossing experiments showed that a range of variation in heading date occurred in the later generations and that the region might contain at least a major gene and two additional recessive genes controlling photoperiod sensitivity. Gene mapping experiments showed that the major gene was Se-1 and that a recessive gene (tentatively named se-pat) was loosely linked to it. The responses to photoperiods were examined among the different genotypes under natural and controlled conditions. The two genes acted additively on the degree of photoperiod sensitivity. However, se-pat plants showed a response to photoperiods that differed from that of the other sensitive lines; a short-day treatment at the seedling stage delayed heading in the former plants, suggesting that the manner of its expression was age-dependent. A recessive gene similar to se-pat seemed to be widely distributed in wild and cultivated rice, suggesting that the gene complex in the region plays a significant role in response to photoperiod. Received: 8 October 1997 / Accepted: 1 April 1998     

Optimization of economic policies and investment projects using a fuzzy logic based cost-effectiveness model of coral reef quality: empirical results for Montego Bay, Jamaica

 For effective mitigation of human impacts, quantitative models are required that facilitate a comprehensive analysis of the effects of human activity on reefs. Fuzzy logic procedures generate a complex dose-response surface that models the relationships among coral abundance and various inputs (e.g., physical damage, sedimentation, nutrient influx), within the context of the abiotic marine environment. This is linked to a nonlinear economic structure incorporating technical interventions (e.g., pollution treatment) and policy interventions (e.g., taxation) in eight economic sectors. Optimization provides insights into the most cost-effective means for protecting coral reefs under different reef quality targets. The research demonstrates that: (1) it is feasible to use fuzzy logic to model complex interactions in coral reef ecosystems; and, (2) conventional economic procedures for modeling cost-effectiveness can result in sub-optimal policy choices when applied to complex systems such as coral reefs. In Montego Bay, Jamaica, up to a 20% increase in coral abundance may be achievable through using appropriate policy measures having a present value cost of US$153 million over 25 years; a 10% increase is achievable at a cost of US$12 million. Accepted: 20 June 1999