The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world

Biotechnology has empirically established that it is easier to construct and evaluate variant genes and proteins than to account for the emergence and function of wild-type macromolecules. Systematizing this constructive approach, synthetic biology now promises to infer and assemble entirely novel genomes, cells and ecosystems. It is argued here that the theoretical and computational tools needed for this endeavor are missing altogether. However, such tools may not be required for diversifying organisms at the basic level of their chemical constitution by adding, substituting or removing elements and molecular components through directed evolution under selection. Most importantly, chemical diversification of life forms could be designed to block metabolic cross-feed and genetic cross-talk between synthetic and wild species and hence protect natural habitats and human health through novel types of containment.     

Synthetic Genomics and Synthetic Biology Applications Between Hopes and Concerns

New organisms and biological systems designed to satisfy human needs are among the aims of synthetic genomics and synthetic biology. Synthetic biology seeks to model and construct biological components, functions and organisms that do not exist in nature or to redesign existing biological systems to perform new functions. Synthetic genomics, on the other hand, encompasses technologies for the generation of chemically-synthesized whole genomes or larger parts of genomes, allowing to simultaneously engineer a myriad of changes to the genetic material of organisms. Engineering complex functions or new organisms in synthetic biology are thus progressively becoming dependent on and converging with synthetic genomics. While applications from both areas have been predicted to offer great benefits by making possible new drugs, renewable chemicals or clean energy, they have also given rise to concerns about new safety, environmental and socio-economic risks – stirring an increasingly polarizing debate. Here we intend to provide an overview on recent progress in biomedical and biotechnological applications of synthetic genomics and synthetic biology as well as on arguments and evidence related to their possible benefits, risks and governance implications.     

Advances in molecular cloning

“Molecular cloning” meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.     

Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite Gateway^TM cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite Gateway^TM cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of “classical construct generation” and allows focusing on construct design.     

Recombination within a Subclass of Restriction Fragment Length Polymorphisms May Help Link Classical and Molecular Genetics

Restriction fragment length polymorphisms (RFLPs) are being used to construct complete linkage maps for many eukaryotic genomes. These RFLP maps can be used to predict the inheritance of important phenotypic loci and will assist in the molecular cloning of linked gene(s) which affect phenotypes of scientific, medical and agronomic importance. However, genetic linkage implies very little about the actual physical distances between loci. An assay is described which uses genetic recombinants to measure physical distance from a DNA probe to linked phenotypic loci. We have defined the subset of all RFLPs which have polymorphic restriction sites at both ends as class II RFLPs. The frequency of class II RFLPs is computed as a function of sequence divergence and total RFLP frequency for highly divergent genomes. Useful frequencies exist between organisms which differ by more than 7% in DNA sequence. Recombination within class II RFLPs will produce fragments of novel sizes which can be assayed by pulsed field electrophoresis to estimate physical distance in kilobase pairs between linked RFLP and phenotypic loci. This proposed assay should have particular applications to crop plants where highly divergent and polymorphic species are often genetically compatible and thus, where class II RFLPs will be most frequent.     

Synthetic biology: new strategies for directing design

The advancement of synthetic biology is thanks, in large part, to continuing improvements in DNA synthesis. The expansion of synthetic biology into the realm of metabolic engineering has shifted the focus from simply making novel synthetic biological parts to answering the question of how we employ these biological parts to construct genomes that ultimately give rise to useful phenotypes. Much like protein engineering, the answer to this will be arrived at following the combination of rational design and evolutionary approaches. This review will highlight some of the new DNA synthesis-enabled search methods and discuss the application of such methods to the creation of synthetic gene networks and genomes.     

pTransgenesis: a cross-species, modular transgenesis resource

As studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.     

Recent advances in the genetics of the clostridia

Several laboratories around the world have started work on genetic analysis of clostridia. Interest in this diverse group of anaerobic organisms has grown with increasing awareness of the benefits that may accrue from their biotechnological exploitation. Research to date has focussed on construction of shuttle vectors containing replicons from clostridial and streptococcal plasmids, development of methods for transferring genes, and molecular cloning of genes--especially those involved in toxigenicity, fermentative metabolism and polysaccharide utilization. In selected species gene transfer by protoplast transformation, electroporation and conjugation has been accomplished and transposable elements have been introduced. It can be anticipated that our understanding of the molecular biology of these interesting organisms will grow rapidly in the future, bringing with it improved prospects for rational biotechnological exploitation.     

New ligation-independent cloning vectors compatible with a high-throughput platform for parallel construct expression evaluation using baculovirus-infected insect cells

Biomedical research has undergone a major shift in emphasis over the past decade from characterizing the genomes of organisms to characterizing their proteomes. The high-throughput approaches that were successfully applied to sequencing of genomes, such as miniaturization and automation, have been adapted for high-throughput cloning and protein production. High-throughput platforms allow for a multi-construct, multi-parallel approach to expression optimization and construct evaluation. We describe here a series of baculovirus transfer and expression vectors that contain ligation-independent cloning regions originally designed for use in high-throughput Escherichia coli expression evaluation. These new vectors allow for parallel cloning of the same gene construct into a variety of baculovirus or E. coli expression vectors. A high-throughput platform for construct expression evaluation in baculovirus-infected insect cells was developed to utilize these vectors. Data from baculovirus infection expression trials for multiple constructs of two target protein systems relevant to the study of human diseases are presented. The target proteins exhibit a wide variation in behavior and illustrate the benefit of investigating multiple cell types, fusion partners and secretion signals in optimization of constructs and conditions for eukaryotic protein production.     

合成生物学技术的研究进展——DNA合成、组装与基因组编辑

合成生物学作为一门新兴学科,其目标主要有两点:一是利用非天然的分子使其出现生命的现象,也就是―人造生命‖;二是―改造生命‖,比如利用一种生命体的元件(或经过人工改造),组装到另一个生命体中,使其产生特定功能。无论是哪种目的,对生命遗传物质DNA的操作都非常关键,其具体包括DNA的从头合成、组装和编辑等。同时,这些使能技术的进步也促进了合成生物学其他领域的发展。本文介绍了DNA操作相关的合成生物学使能技术的最新进展。     

Improved method for constructing plant amiRNA vectors with blue-white screening and MAGIC

Artificial microRNA (amiRNA) technology is a novel tool in reverse genetic research for discovering or validating gene functions in plants. A convenient cloning strategy has been developed to construct plant amiRNA vectors based on lacO reconstruction and mating-assisted, genetically-integrated cloning (MAGIC). The amiRNA precursor fragment was generated by PCR and inserted into a small donor plasmid through reconstruction of integrated lacO sequence. Blue recombinants were selected on plates containing X-gal and the efficiency of successful clones was 100%. The amiRNA expression cassette was transferred from the donor plasmid to the recipient plasmid p1301-gfp through MAGIC and an amiRNA expression plasmid was created. More than 40 plant amiRNA vectors were generated through this method, one of which was transformed into Arabidopsis thaliana and the target gene was silenced efficiently. The approach will be useful for amiRNA expression vectors construction in plants.     

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.     

SpecHap: a diploid phasing algorithm based on spectral graph theory

Haplotype phasing plays an important role in understanding the genetic data of diploid eukaryotic organisms. Different sequencing technologies (such as next-generation sequencing or third-generation sequencing) produce various genetic data that require haplotype assembly. Although multiple diploid haplotype phasing algorithms exist, only a few will work equally well across all sequencing technologies. In this work, we propose SpecHap, a novel haplotype assembly tool that leverages spectral graph theory. On both in silico and whole-genome sequencing datasets, SpecHap consumed less memory and required less CPU time, yet achieved comparable accuracy with state-of-art methods across all the test instances, which comprises sequencing data from next-generation sequencing, linked-reads, high-throughput chromosome conformation capture, PacBio single-molecule real-time, and Oxford Nanopore long-reads. Furthermore, SpecHap successfully phased an individual Ambystoma mexicanum, a species with gigantic diploid genomes, within 6 CPU hours and 945MB peak memory usage, while other tools failed to yield results either due to memory overflow (40GB) or time limit exceeded (5 days). Our results demonstrated that SpecHap is scalable, efficient, and accurate for diploid phasing across many sequencing platforms.     

Recent advances in the genetics of the clostridia

Abstract Several laboratories around the world have started work on genetic analysis of clostridia. Interest in this diverse group of anaerobic organisms has grown with increasing awareness of the benefits that may accrue from their biotechnological exploitation. Research to date has focussed on construction of shuttle vectors containing replicons from clostridial and streptococcal plasmids, development of methods for transferring genes, and molecular cloning of genes—especially those involved in toxigenicity, fermentative metabolism and polysaccharide utilization. In selected species gene transfer by protoplast transformation, electroporation and conjugation has been accomplished and transposable elements have been introduced. It can be anticipated that our understanding of the molecular biology of these interesting organisms will grow rapidly in the future, bringing with it improved prospects for rational biotechnological exploitation.     

Defining the roles of cis-acting RNA elements in tombusvirus replicase assembly in vitro

In addition to its central role as a template for replication and translation, the viral plus-strand RNA genome also has nontemplate functions, such as recruitment to the site of replication and assembly of the viral replicase, activities that are mediated by cis-acting RNA elements within viral genomes. Two noncontiguous RNA elements, RII(+)-SL (located internally in the tombusvirus genome) and RIV (located at the 3'-terminus), are involved in template recruitment into replication and replicase assembly; however, the importance of each of these RNA elements for these two distinct functions is not fully elucidated. We used an in vitro replicase assembly assay based on yeast cell extract and purified recombinant tombusvirus replication proteins to show that RII(+)-SL, in addition to its known requirement for recruitment of the plus-strand RNA into replication, is also necessary for assembly of an active viral replicase complex. Additional studies using a novel two-component RNA system revealed that the recruitment function of RII(+)-SL can be provided in trans by a separate RNA and that the replication silencer element, located within RIV, defines the template that is used for initiation of minus-strand synthesis. Collectively, this work has revealed new functions for tombusvirus cis-acting RNA elements and provided insights into the pioneering round of minus-strand synthesis.     

Available methods for assembling expression cassettes for synthetic biology

Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.     

The expression and characterization of rat IgE produced by construction of the epsilon-heavy chain gene from exon modules.

A system of exon "modules" was produced from the functionally rearranged epsilon-heavy gene isolated from the rat IgE-secreting immunocytoma IR162. The five individual exons, encoding the variable and constant region domains, were isolated and subcloned into the multiple cloning site of a pair of plasmid vectors with opposed orientation multiple cloning sites. The use of opposed orientation multiple cloning sites and the flanking restriction enzyme sites contained therein allows for the modular manipulation of the gene. These exon modules were initially used to reconstruct the epsilon-heavy chain gene into the native configuration to demonstrate the efficacy of the modular system for synthesis of IgE. Upon transfection into the rat myeloma cell line Y3, the reconstructed gene produced a polypeptide that associated with the endogenous light chain polypeptide and was secreted from the cell as tetrameric IgE. All physical and functional characterizations indicate that the IgE molecule produced is indistinguishable from native IR162 IgE. This modular system of exons will facilitate the manipulation of IgE structure through the systematic assembly of different epsilon-heavy chain mutant constructions. The resulting novel IgE proteins will be very useful to study the molecular nature of the interaction of IgE with its Fc receptor.     

Prokaryotic gene clusters: a rich toolbox for synthetic biology

Bacteria construct elaborate nanostructures, obtain nutrients and energy from diverse sources, synthesize complex molecules, and implement signal processing to react to their environment. These complex phenotypes require the coordinated action of multiple genes, which are often encoded in a contiguous region of the genome, referred to as a gene cluster. Gene clusters sometimes contain all of the genes necessary and sufficient for a particular function. As an evolutionary mechanism, gene clusters facilitate the horizontal transfer of the complete function between species. Here, we review recent work on a number of clusters whose functions are relevant to biotechnology. Engineering these clusters has been hindered by their regulatory complexity, the need to balance the expression of many genes, and a lack of tools to design and manipulate DNA at this scale. Advances in synthetic biology will enable the large-scale bottom-up engineering of the clusters to optimize their functions, wake up cryptic clusters, or to transfer them between organisms. Understanding and manipulating gene clusters will move towards an era of genome engineering, where multiple functions can be "mixed-and-matched" to create a designer organism.