Synthetic biology regulation in Europe: containment, release and beyond.

While synthetic biology is hoped to hold promise and potential to address pressing global challenges, the issue of regulation is an under-appreciated challenge. Particularly in Europe, the regulatory frameworks involved are rooted in historical concepts based on containment and release. Through a series of case studies including a field-use biosensor intended to detect arsenic in well water in Nepal and Bangladesh, and insects engineered for sterility, we explore the implications that this regulatory and conceptual divide has had on the deployment of synthetic biology projects in different national contexts. We then consider some of the broader impacts that regulation can have on the development of synthetic biology as a field, not only in Europe but also globally, with a particular emphasis on low- and middle-income countries. We propose that future regulatory adaptability would be increased by moving away from a containment and release dichotomy and toward a more comprehensive assessment that accounts for the possibility of varying degrees of 'contained release'. Graphical Abstract.

T7 RNA polymerase-driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis.

The majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr-ts-1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome-encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase-driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis.

Empirical Bayes method for reducing false discovery rates of correlation matrices with block diagonal structure.

BACKGROUND: Correlation matrices are important in inferring relationships and networks between regulatory or signalling elements in biological systems. With currently available technology sample sizes for experiments are typically small, meaning that these correlations can be difficult to estimate. At a genome-wide scale estimation of correlation matrices can also be computationally demanding. RESULTS: We develop an empirical Bayes approach to improve covariance estimates for gene expression, where we assume the covariance matrix takes a block diagonal form. Our method shows lower false discovery rates than existing methods on simulated data. Applied to a real data set from Bacillus subtilis we demonstrate it's ability to detecting known regulatory units and interactions between them. CONCLUSIONS: We demonstrate that, compared to existing methods, our method is able to find significant covariances and also to control false discovery rates, even when the sample size is small (n=10). The method can be used to find potential regulatory networks, and it may also be used as a pre-processing step for methods that calculate, for example, partial correlations, so enabling the inference of the causal and hierarchical structure of the networks.

High molecular weight DNA assembly in vivo for synthetic biology applications.

DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis.

The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis.

Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome.

BACKGROUND: Escherichia coli K-12 is a frequently used host for a number of synthetic biology and biotechnology applications and chassis for the development of the minimal cell factories. Novel approaches for integrating high molecular weight DNA into the E. coli chromosome would therefore greatly facilitate engineering efforts in this bacterium. RESULTS: We developed a reliable and flexible lambda Red recombinase-based system, which utilizes overlapping DNA fragments for integration of the high molecular weight DNA into the E. coli chromosome. Our chromosomal integration strategy can be used to integrate high molecular weight DNA of variable length into any non-essential locus in the E. coli chromosome. Using this approach we integrated 15 kb DNA encoding sucrose catabolism and lactose metabolism and transport operons into the fliK locus of the flagellar region 3b in the E. coli K12 MG1655 chromosome. Furthermore, with this system we integrated 50 kb of Bacillus subtilis 168 DNA into two target sites in the E. coli K12 MG1655 chromosome. The chromosomal integrations into the fliK locus occurred with high efficiency, inhibited motility, and did not have a negative effect on the growth of E. coli. CONCLUSIONS: In addition to the rational design of synthetic biology devices, our high molecular weight DNA chromosomal integration system will facilitate metabolic and genome-scale engineering of E. coli.

Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.

Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes.

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.

Characterization of Intrinsic Properties of Promoters.

Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic contexts of the measurement. Furthermore, in vivo measurement methods must accommodate variation in translation, protein folding, and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered to be extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents, and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method to quantitatively describe promoter properties.

Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions.

The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits.

Identification and validation of novel chromosomal integration and expression loci in Escherichia coli flagellar region 1.

Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci.

NextGen sequencing reveals short double crossovers contribute disproportionately to genetic diversity in Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is a widespread protozoan parasite of animals that causes zoonotic disease in humans. Three clonal variants predominate in North America and Europe, while South American strains are genetically diverse, and undergo more frequent recombination. All three northern clonal variants share a monomorphic version of chromosome Ia (ChrIa), which is also found in unrelated, but successful southern lineages. Although this pattern could reflect a selective advantage, it might also arise from non-Mendelian segregation during meiosis. To understand the inheritance of ChrIa, we performed a genetic cross between the northern clonal type 2 ME49 strain and a divergent southern type 10 strain called VAND, which harbors a divergent ChrIa. RESULTS: NextGen sequencing of haploid F1 progeny was used to generate a genetic map revealing a low level of conventional recombination, with an unexpectedly high frequency of short, double crossovers. Notably, both the monomorphic and divergent versions of ChrIa were isolated with equal frequency. As well, ChrIa showed no evidence of being a sex chromosome, of harboring an inversion, or distorting patterns of segregation. Although VAND was unable to self fertilize in the cat, it underwent successful out-crossing with ME49 and hybrid survival was strongly associated with inheritance of ChrIII from ME49 and ChrIb from VAND. CONCLUSIONS: Our findings suggest that the successful spread of the monomorphic ChrIa in the wild has not been driven by meiotic drive or related processes, but rather is due to a fitness advantage. As well, the high frequency of short double crossovers is expected to greatly increase genetic diversity among progeny from genetic crosses, thereby providing an unexpected and likely important source of diversity.

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages.

Marked phenotypic variation characterizes isolates of Toxoplasma gondii, a ubiquitous zoonotic parasite that serves as an important experimental model for studying apicomplexan parasites. Progress in identifying the heritable basis for clinically and epidemiologically significant differences requires a robust system for describing and interpreting evolutionary subdivisions in this prevalent pathogen. To develop such a system, we have examined more than 950 isolates collected from around the world and genotyped them using three independent sets of polymorphic DNA markers, sampling 30 loci distributed across all nuclear chromosomes as well as the plastid genome. Our studies reveal a biphasic pattern consisting of regions in the Northern Hemisphere where a few, highly clonal and abundant lineages predominate; elsewhere, and especially in portions of South America are characterized by a diverse assemblage of less common genotypes that show greater evidence of recombination. Clustering methods were used to organize the marked genetic diversity of 138 unique genotypes into 15 haplogroups that collectively define six major clades. Analysis of gene flow indicates that a small number of ancestral lineages gave rise to the existing diversity through a process of limited admixture. Identification of reference strains for these major groups should facilitate future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission.

A monomorphic haplotype of chromosome Ia is associated with widespread success in clonal and nonclonal populations of Toxoplasma gondii.

UNLABELLED: Toxoplasma gondii is a common parasite of animals that also causes a zoonotic infection in humans. Previous studies have revealed a strongly clonal population structure that is shared between North America and Europe, while South American strains show greater genetic diversity and evidence of sexual recombination. The common inheritance of a monomorphic version of chromosome Ia (referred to as ChrIa*) among three clonal lineages from North America and Europe suggests that inheritance of this chromosome might underlie their recent clonal expansion. To further examine the diversity and distribution of ChrIa, we have analyzed additional strains with greater geographic diversity. Our findings reveal that the same haplotype of ChrIa* is found in the clonal lineages from North America and Europe and in older lineages in South America, where sexual recombination is more common. Although lineages from all three continents harbor the same conserved ChrIa* haplotype, strains from North America and Europe are genetically separate from those in South America, and these respective geographic regions show limited evidence of recent mixing. Genome-wide, array-based profiling of polymorphisms provided evidence for an ancestral flow from particular older southern lineages that gave rise to the clonal lineages now dominant in the north. Collectively, these data indicate that ChrIa* is widespread among nonclonal strains in South America and has more recently been associated with clonal expansion of specific lineages in North America and Europe. These findings have significant implications for the spread of genetic loci influencing transmission and virulence in pathogen populations. IMPORTANCE: Understanding parasite population structure is important for evaluating the potential spread of pathogenicity determinants between different geographic regions. Examining the genetic makeup of different isolates of Toxoplasma gondii from around the world revealed that chromosome Ia is highly homogeneous among lineages that predominate on different continents and within genomes that were otherwise quite divergent. This pattern of recent shared ancestry is highly unusual and suggests that some gene(s) found on this chromosome imparts an unusual fitness advantage that has resulted in its recent spread. Although the basis for the conservation of this particularly homogeneous chromosome is unknown, it may have implications for the transmission of infection and spread of human disease.

Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America.

Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Previous studies have revealed a strongly clonal population structure in North America and Europe, while strains from South America are genetically separate and more diverse. However, the composition within North America has been questioned by recent descriptions of genetically more variable strains from this region. Here, we examined an expanded set of isolates using sequenced-based phylogenetic and population analyses to re-evaluate the population structure of T. gondii in North America. Our findings reveal that isolates previously defined by atypical restriction fragment length polymorphism patterns fall into two discrete groups. In one case, these new isolates represent variants of an existing lineage, from which they differ only by minor mutational drift. However, in the second case, it is evident that these isolates define a completely new lineage that is common in North America. Support for this new lineage was based on phylogeny, principle components analysis, STRUCTURE analyses, and statistical analysis of gene flow between groups. This new group, referred to as haplogroup 12, contains divergent genotypes previously referred to as A and X, isolated from sea otters. Consistent with this, group 12 was found primarily in wild animals, as well as occasionally in humans. This new lineage also has a highly clonal population structure. Analysis of the inheritance of multilocus genotypes revealed that different strains within group 12 are the products of a single recombination event between type 2 and a unique parental lineage. Collectively, the archetypal type 2 has been associated with clonal expansion of a small number of lineages in the North, as a consequence of separate but infrequent genetic crosses with several different parental lines.

Genetic diversity of Toxoplasma gondii in animals and humans.

Toxoplasma gondii is one of the most widespread parasites of domestic, wild, and companion animals, and it also commonly infects humans. Toxoplasma gondii has a complex life cycle. Sexual development occurs only in the cat gut, while asexual replication occurs in many vertebrate hosts. These features combine to create an unusual population structure. The vast majority of strains in North America and Europe fall into three recently derived, clonal lineages known as types I, II and III. Recent studies have revealed that South American strains are more genetically diverse and comprise distinct genotypes. These differences have been shaped by infrequent sexual recombination, population sweeps and biogeography. The majority of human infections that have been studied in North America and Europe are caused by type II strains, which are also common in agricultural animals from these regions. In contrast, several diverse genotypes of T. gondii are associated with severe infections in humans in South America. Defining the population structure of T. gondii from new regions has important implications for transmission, immunogenicity and pathogenesis.

Selection at a single locus leads to widespread expansion of Toxoplasma gondii lineages that are virulent in mice.

Pathogenicity differences among laboratory isolates of the dominant clonal North American and European lineages of Toxoplasma gondii are largely controlled by polymorphisms and expression differences in rhoptry secretory proteins (ROPs). However, the extent to which such differences control virulence in natural isolates of T. gondii, including those from more diverse genetic backgrounds, is uncertain. We elucidated the evolutionary history and functional consequences of diversification in the serine/threonine kinase ROP18, a major virulence determinant in the mouse model. We characterized the extent of sequence polymorphism and the evolutionary forces acting on ROP18 and several antigen-encoding genes within a large collection of natural isolates, comparing them to housekeeping genes and introns. Surprisingly, despite substantial genetic diversity between lineages, we identified just three principal alleles of ROP18, which had very ancient ancestry compared to other sampled loci. Expression and allelic differences between these three alleles of ROP18 accounted for much of the variation in acute mouse virulence among natural isolates. While the avirulent type III allele was the most ancient, intermediate virulent (type II) and highly virulent (type I) lineages predominated and showed evidence of strong selective pressure. Out-group comparison indicated that historical loss of an upstream regulatory element increased ROP18 expression, exposing it to newfound diversifying selection, resulting in greatly enhanced virulence in the mouse model and expansion of new lineages. Population sweeps are evident in many genomes, yet their causes and evolutionary histories are rarely known. Our results establish that up-regulation of expression and selection at ROP18 in T. gondii has resulted in three distinct alleles with widely different levels of acute virulence in the mouse model. Preservation of all three alleles in the wild indicates they are likely adaptations for different niches. Our findings demonstrate that sweeping changes in population structure can result from alterations in a single gene.

The early years of Toxoplasma research: What's past is prologue.

In the century since the first description of Toxoplasma gondii history and circumstance have led scientists to define this organism in diverse contexts. From its discovery by researchers shaped by early 20th century versions of the germ theory to its more recent roles as an important globally distributed pathogen and a model apicomplexan, our definitions of Toxoplasma are as much a reflection of our frame of reference as they are an absolute definition of this organism. Although these transformations act as portals for new avenues of investigation, the essential questions that inform current research are founded in the work of early investigators who studied Toxoplasma.