Genomic markers on synthetic genomes

Genome synthesis endows scientists the ability of de novo creating genomes absent in nature, by thorough redesigning DNA sequences and introducing numerous custom features. However, the genome synthesis is a labor‐ and time‐consuming work, and thus it is a challenge to verify and quantify the synthetic genome rapidly and precisely. Thus, specific DNA sequences different from native genomic sequences are designed into synthetic genomes during synthesis, namely genomic markers. Genomic markers can be easily detected by PCR reaction, whole‐genome sequencing (WGS) and a variety of methods to identify the synthetic genome from native one. Here, we review types and applications of genomic markers utilized in synthetic genomes, with the hope of providing a guidance for future works.     

Synthetic genome engineering forging new frontiers for wine yeast

Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiae became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project – the Synthetic Yeast Genome (Sc2.0) Project – is now underway to synthesize all 16 chromosomes (～12?Mb carrying ～6000 genes) of the sequenced S288c laboratory strain by 2018. If successful, S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future holds is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast strain (AWRI1631), which was recently achieved via metabolic pathway engineering and synthetic enzyme fusion. A peek over the horizon is revealing that the future of “Wine Yeast 2.0” is already here. Therefore, this article seeks to help prepare the wine industry – an industry rich in history and tradition on the one hand, and innovation on the other – for the inevitable intersection of the ancient art practiced by winemakers and the inventive science of pioneering “synthetic genomicists”. It would be prudent to proactively engage all stakeholders – researchers, industry practitioners, policymakers, regulators, commentators, and consumers – in a meaningful dialog about the potential challenges and opportunities emanating from Synthetic Biology. To capitalize on the new vistas of synthetic yeast genomics, this paper presents wine yeast research in a fresh context, raises important questions and proposes new directions.     

Characterization of the core and accessory genomes of Pseudomonas aeruginosa using bioinformatic tools Spine and AGEnt

Background

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for many infections in hospitalized and immunocompromised patients. Previous reports estimated that approximately 10% of its 6.6 Mbp genome varies from strain to strain and is therefore referred to as “accessory genome”. Elements within the accessory genome of P. aeruginosa have been associated with differences in virulence and antibiotic resistance. As whole genome sequencing of bacterial strains becomes more widespread and cost-effective, methods to quickly and reliably identify accessory genomic elements in newly sequenced P. aeruginosa genomes will be needed.

Results

We developed a bioinformatic method for identifying the accessory genome of P. aeruginosa. First, the core genome was determined based on sequence conserved among the completed genomes of twelve reference strains using Spine, a software program developed for this purpose. The core genome was 5.84 Mbp in size and contained 5,316 coding sequences. We then developed an in silico genome subtraction program named AGEnt to filter out core genomic sequences from P. aeruginosa whole genomes to identify accessory genomic sequences of these reference strains. This analysis determined that the accessory genome of P. aeruginosa ranged from 6.9-18.0% of the total genome, was enriched for genes associated with mobile elements, and was comprised of a majority of genes with unknown or unclear function. Using these genomes, we showed that AGEnt performed well compared to other publically available programs designed to detect accessory genomic elements. We then demonstrated the utility of the AGEnt program by applying it to the draft genomes of two previously unsequenced P. aeruginosa strains, PA99 and PA103.

Conclusions

The P. aeruginosa genome is rich in accessory genetic material. The AGEnt program accurately identified the accessory genomes of newly sequenced P. aeruginosa strains, even when draft genomes were used. As P. aeruginosa genomes become available at an increasingly rapid pace, this program will be useful in cataloging the expanding accessory genome of this bacterium and in discerning correlations between phenotype and accessory genome makeup. The combination of Spine and AGEnt should be useful in defining the accessory genomes of other bacterial species as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-737) contains supplementary material, which is available to authorized users.     

Proteomic Detection of Non-Annotated Protein-Coding Genes in Pseudomonas fluorescens Pf0-1

Genome sequences are annotated by computational prediction of coding sequences, followed by similarity searches such as BLAST, which provide a layer of possible functional information. While the existence of processes such as alternative splicing complicates matters for eukaryote genomes, the view of bacterial genomes as a linear series of closely spaced genes leads to the assumption that computational annotations that predict such arrangements completely describe the coding capacity of bacterial genomes. We undertook a proteomic study to identify proteins expressed by Pseudomonas fluorescens Pf0-1 from genes that were not predicted during the genome annotation. Mapping peptides to the Pf0-1 genome sequence identified sixteen non-annotated protein-coding regions, of which nine were antisense to predicted genes, six were intergenic, and one read in the same direction as an annotated gene but in a different frame. The expression of all but one of the newly discovered genes was verified by RT-PCR. Few clues as to the function of the new genes were gleaned from informatic analyses, but potential orthologs in other Pseudomonas genomes were identified for eight of the new genes. The 16 newly identified genes improve the quality of the Pf0-1 genome annotation, and the detection of antisense protein-coding genes indicates the under-appreciated complexity of bacterial genome organization.     

Isolation, propagation and characterization of replication requirements of reiteration mutants of Simian Virus 40.

We have identified five reiteration mutants from serially-propagated, defective stocks of Simian Virus 40 and DAR virus (an SV40³ variant of human origin). The genomes of these mutants contain tandem repeats of specific segments of the SV40 genome. In order to propagate individual reiteration mutants, the monomer DNA segments from the mutant genomes are separated from wild-type SV40 DNA after cleavage by certain bacterial restriction endonucleases which produce short cohesive termini at their cleavage sites. These monomer segments, which are one-third, one-fourth, or one-fifth the size of wild-type SV40 DNA, are then circularized in vitro using bacteriophage T4 polynucleotide ligase and used to infect African green monkey kidney cells in the presence of wild-type or temperature-sensitive mutant DNAs as helpers. While wild-type SV40 and late temperature-sensitive mutants can serve as helpers in the replication and amplification of these minicircular DNAs, early temperature-sensitive mutant genomes are unable to do so at the nonpermissive temperature. The minicircular DNAs are amplified in vivo through an arithmetic series of oligomers. Encapsidation of reiterated molecules between 70 and 100% the size of wild-type SV40 DNA is observed, although reiterated viral DNA molecules much larger than unit size are formed in vivo.     

Genome Writing: Current Progress and Related Applications

The ultimate goal of synthetic biology is to build customized cells or organisms to meet specific industrial or medical needs. The most important part of the customized cell is a synthetic genome. Advanced genomic writing technologies are required to build such an artificial genome. Recently, the partially-completed synthetic yeast genome project represents a milestone in this field. In this mini review, we briefly introduce the techniques for de novo genome synthesis and genome editing. Furthermore, we summarize recent research progresses and highlight several applications in the synthetic genome field. Finally, we discuss current challenges and future prospects.     

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases

Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.     

Whole-genome synthesis and characterization of viable S13-like bacteriophages

Background

Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template.

Methodology/Principal Findings

The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl₂, the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition.

Conclusions/Significance

The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.     

Defining the Estimated Core Genome of Bacterial Populations Using a Bayesian Decision Model

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance.     

Teaching Synthetic Biology, Bioinformatics and Engineering to Undergraduates: The Interdisciplinary Build-a-Genome Course

A major challenge in undergraduate life science curricula is the continual evaluation and development of courses that reflect the constantly shifting face of contemporary biological research. Synthetic biology offers an excellent framework within which students may participate in cutting-edge interdisciplinary research and is therefore an attractive addition to the undergraduate biology curriculum. This new discipline offers the promise of a deeper understanding of gene function, gene order, and chromosome structure through the de novo synthesis of genetic information, much as synthetic approaches informed organic chemistry. While considerable progress has been achieved in the synthesis of entire viral and prokaryotic genomes, fabrication of eukaryotic genomes requires synthesis on a scale that is orders of magnitude higher. These high-throughput but labor-intensive projects serve as an ideal way to introduce undergraduates to hands-on synthetic biology research. We are pursuing synthesis of Saccharomyces cerevisiae chromosomes in an undergraduate laboratory setting, the Build-a-Genome course, thereby exposing students to the engineering of biology on a genomewide scale while focusing on a limited region of the genome. A synthetic chromosome III sequence was designed, ordered from commercial suppliers in the form of oligonucleotides, and subsequently assembled by students into ~750-bp fragments. Once trained in assembly of such DNA “building blocks” by PCR, the students accomplish high-yield gene synthesis, becoming not only technically proficient but also constructively critical and capable of adapting their protocols as independent researchers. Regular “lab meeting” sessions help prepare them for future roles in laboratory science.     

Rep-PCR Identifies Both Inter- and Intra-Specific Mitochondrial Genome Differences in Carthamus

Mitochondria are derived from ancient prokaryotic endosymbionts, and their genomes exhibit similarities to prokaryote genomes. Therefore, it was hypothesized that the molecular techniques suitable for distinguishing prokaryotic genomes could also be used to assess mitochondrial diversity. The rep-PCR (repetitive element palindromic-PCR) technique, based on the repetitive sequences found in bacterial genomes, has been used extensively for identifying and distinguishing bacterial strains. This study was undertaken to evaluate the utility of rep-PCR for identifying mitochondrial (mt) genome diversity in safflower (Carthamus tinctorius L.) and its wild relatives. Using three sets of commonly used primers, BOX, ERIC and REP, both inter-specific and intra-specific mt genome diversities in Carthamus were identified. To confirm that the amplicons obtained with rep-PCR were derived from mitochondrial genomes, we cloned and sequenced six randomly chosen bands from rep-PCR gels and demonstrated that the amplified products were mitochondrial-genome-specific. The advantages of rep-PCR in assessing chondriome variability are discussed.     

Whole genome engineering by synthesis

Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GP-write, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.     

Autophagy determines mtDNA copy number dynamics during starvation

Derived from bacterial ancestors, mitochondria have maintained their own albeit strongly reduced genome, mitochondrial DNA (mtDNA), which encodes for a small and highly specialized set of genes. MtDNA exists in tens to thousands of copies packaged in numerous nucleoprotein complexes, termed nucleoids, distributed throughout the dynamic mitochondrial network. Our understanding of the mechanisms of how cells regulate the copy number of mitochondrial genomes has been limited. Here, we summarize and discuss our recent findings that Mip1/POLG (mitochondrial DNA polymerase gamma) critically controls mtDNA copy number by operating in 2 opposing modes, synthesis and, unexpectedly, degradation of mtDNA, when yeast cells face nutrient starvation. The balance of the 2 modes of Mip1/POLG and thus mtDNA copy number dynamics depends on the integrity of macroautophagy/autophagy, which sustains continuous synthesis and maintenance of mtDNA. In autophagy-deficient cells, a combination of nucleotide insufficiency and elevated mitochondrial ROS production impairs mtDNA synthesis and drives mtDNA degradation by the 3?-5?-exonuclease activity of Mip1/POLG resulting in mitochondrial genome depletion and irreversible respiratory deficiency.

Abbrivations: mtDNA: mitochondrial DNA; mtDCN: mitochondrial DNA copy number.     

Recognizing the pseudogenes in bacterial genomes

Pseudogenes are now known to be a regular feature of bacterial genomes and are found in particularly high numbers within the genomes of recently emerged bacterial pathogens. As most pseudogenes are recognized by sequence alignments, we use newly available genomic sequences to identify the pseudogenes in 11 genomes from 4 bacterial genera, each of which contains at least 1 human pathogen. The numbers of pseudogenes range from 27 in Staphylococcus aureus MW2 to 337 in Yersinia pestis CO92 (e.g. 1–8% of the annotated genes in the genome). Most pseudogenes are formed by small frameshifting indels, but because stop codons are A + T-rich, the two low-G + C Gram-positive taxa (Streptococcus and Staphylococcus) have relatively high fractions of pseudogenes generated by nonsense mutations when compared with more G + C-rich genomes. Over half of the pseudogenes are produced from genes whose original functions were annotated as ‘hypothetical’ or ‘unknown’; however, several broadly distributed genes involved in nucleotide processing, repair or replication have become pseudogenes in one of the sequenced Vibrio vulnificus genomes. Although many of our comparisons involved closely related strains with broadly overlapping gene inventories, each genome contains a largely unique set of pseudogenes, suggesting that pseudogenes are formed and eliminated relatively rapidly from most bacterial genomes.     

Microbial genescapes: a prokaryotic view of the yeast genome

We examine the translated open reading frames (ORFs) of the yeast Saccharomyces cerevisiae, focusing on those that have FASTA matches in phyletically defined sets of completely sequenced genomes. On this basis, we identify archaeal yeast, bacterial yeast, universal yeast, and yeast ORFs that do not have a match in any of nine prokaryote genomes. Similarly, we examine the yeast mitochondrial genome and the subset of the yeast nuclear ORFs identified as being involved in mitochondrial biogenesis. For the yeast ORFs that match one or more ORFs in these prokaryote genomes, we examine the phyletic and functional distributions of these matches as a function of match strength. These results provide genome level insights into the origin of the eukaryotic cell and the origin of mitochondria. More generally, they exemplify how the growing database of prokaryote genome sequences can help us understand eukaryote genomes.