Medium Copy Number Research Articles

A mix-and-match reverse genetics system for evaluating genetic determinants of orthobunyavirus neurological disease.

The encephalitic orthobunyaviruses have tri-segmented, negative sense RNA genomes and can cause severe neurological disease in humans, including La Crosse virus (LACV), which is the leading cause of pediatric arboviral encephalitis in the United States. However, little is known about the genetic factors that drive neuropathogenesis. Reverse genetics systems (RGS) are valuable tools for studying viral genetics and pathogenesis. Plasmid-based cDNA reverse genetics systems are available for LACV, however the plasmid backbones are medium-copy number and have a propensity for recombination. We therefore generated a plasmid-based cDNA reverse genetics system for LACV utilizing a more stable and high-copy number plasmid backbone. Additionally, we created the first full reverse genetics systems for two closely related orthobunyaviruses, Jamestown Canyon virus (JCV), and Inkoo virus (INKV), which have differing reported disease incidences in humans and differing neuropathogenic phenotypes in mice compared to LACV. We compared wild type (wt) viruses with RGS-derived wt viruses in human neuronal cells and in mice, and found that RGS-derived wt viruses maintained the replication and neuropathogenic phenotypes of their wt counterpart. Additionally, we demonstrated that reverse genetics plasmids from different parental viruses can be readily mixed-and-matched to generate reassortant viruses. This system provides a valuable genetic tool utilizing viruses with differing neuropathogenic phenotypes to investigate the genetic determinants of orthobunyavirus neuropathogenesis.

Comprehensive evaluation and guidance of structural variation detection tools in chicken whole genome sequence data

BackgroundStructural variations (SVs) are widespread across genome and have a great impact on evolution, disease, and phenotypic diversity. Despite the development of numerous bioinformatic tools, commonly referred to as SV callers, tailored for detecting SVs using whole genome sequence (WGS) data and employing diverse algorithms, their performance necessitates rigorous evaluation with real data and validated SVs. Moreover, a considerable proportion of these tools have been primarily designed and optimized using human genome data. Consequently, their applicability and performance in Avian species, characterized by smaller genomes and distinct genomic architectures, remain inadequately assessed.ResultsWe performed a comprehensive assessment of the performance of ten widely used SV callers using population-level real genomic data with the validated five common types of SVs. The performance of SV callers varies with the types and sizes of SVs. As compared with other tools, GRIDSS, Lumpy, Wham, and Manta present better detection accuracy. Pindel can detect more small SVs than others. CNVnator and CNVkit can detect more medium and large copy number variations. Given the poor consistency among different SV callers, the combination calling strategy is not recommended. All tools show poor ability in the detection of insertions (especially with size > 150 bp). At least 50× read depth is required to detect more than 80% of the SVs for most tools.ConclusionsThis study highlights the importance and necessity of using real sequencing data, rather than simulated data only, with validated SVs for SV caller evaluation. Some practical guidance and suggestions are provided for SV detection in future researches.

Prevalence of high-penetrant copy number variants in 7734 low-risk pregnancies

Prevalence of high-penetrant copy number variants in 7734 low-risk pregnancies

Atypical low-copy number plasmid segregation systems, all in one?

Atypical low-copy number plasmid segregation systems, all in one?

Towards synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression

BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

Evaluation of D-loop hypervariable region I variations, haplogroups and copy number of mitochondrial DNA in Bangladeshi population with type 2 diabetes

Evaluation of D-loop hypervariable region I variations, haplogroups and copy number of mitochondrial DNA in Bangladeshi population with type 2 diabetes

Construction and optimization of squalene epoxide synthetic pathway in Escherichia coli

Construction and optimization of squalene epoxide synthetic pathway in Escherichia coli

A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria

Genetically encoded logic gates, especially inverters-NOT gates-are the building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium, and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parametrized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described inverter package (20 different versions of 12 distinct gates) borne by BHR plasmids thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations

Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations

Construction and Characterization of a Medium Copy Number Expression Vector Carrying Auto-Inducible dps Promoter to Overproduce a Bacterial Superoxide Dismutase in Escherichia coli.

Medium copy number expression vector and auto-inducible promoter could be a solution for producing recombinant therapeutic proteins in industrial scale regarding plasmid stability, cost, and product quality. This work aimed to construct a medium copy number pBR322-based expression vector carrying auto-inducible promoter, determine its ability to express heterologous gene, and study its segregational stability. Three stationary-phase promoters of Escherichia coli genes (gadA, dps and sbmC) were used to produce a superoxide dismutase from Staphylococcus equorum (rMnSODSeq) coding region from pBR322Δtet (pBR322-mini). Four plasmids were constructed with different promoters, i.e., T7 (pBMsod), gadA (pMCDsod), dps (pCADsod), and sbmC (pCDSsod) using pBR322-mini as backbone. Results showed that rMnSODSeq expression from pBMsod was significantly higher than that from pJExpress414sod (high copy number plasmid). Meanwhile, rMnSODSeq from pCADsod (auto-inducible promoter) was as high as from pBMsod (IPTG-inducible T7 promoter). rMnSODSeq expressed from pCADsod when bacterial cells entered stationary phase appeared as an active protein band of 23.5kDa when analyzed by zymography and SDS-PAGE. pCADsod displayed the highest stability compared with pBMsod and pJEXpress414sod by plasmid retention assay. We demonstrate the use of an auto-inducible dps promoter to express high level of heterologous protein, an SOD of S. equorum, from a stable expression vector with medium copy number.

A Bacterial Expression Vector Archive (BEVA) for Flexible Modular Assembly of Golden Gate-Compatible Vectors.

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I, and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system, three example vectors were constructed and tested. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins.

Suppression of phenotype of Escherichia coli mutant defective in farnesyl diphosphate synthase by overexpression of gene for octaprenyl diphosphate synthase.

We investigated suppression of the slow growth of an Escherichia coli ispA null mutant lacking farnesyl diphosphate (FPP) synthase (i.e. IspA) by plasmids carrying prenyl diphosphate synthase genes. The growth rates of ispA mutant-transformants harboring a medium-copy number plasmid that carries ispA or ispB were almost the same as that of the wild-type strain. Although the level of FPP in the transformant with the ispA plasmid was almost the same as that in the wild-type strain, the level in the transformant with the ispB plasmid was as low as that in the ispA mutant. Purified octaprenyl diphosphate synthase (IspB) could condense isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form octaprenyl diphosphate and nonaprenyl diphosphate. It is possible that suppression of the slow growth of the ispA mutant by ispB was due to condensation of IPP not only with FPP but also with DMAPP by octaprenyl diphosphate synthase.

Identification and sequence analysis of two novel cryptic plasmids isolated from the vaginal mucosa of South African women

Identification and sequence analysis of two novel cryptic plasmids isolated from the vaginal mucosa of South African women

Orthogonality and Burdens of Heterologous AND Gate Gene Circuits in E.coli.

Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host’s physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behavior of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guidance for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host.

Development of live attenuated bacterial vaccines targeting Escherichia coli heat-labile and heat-stable enterotoxins

Development of live attenuated bacterial vaccines targeting Escherichia coli heat-labile and heat-stable enterotoxins

Refinement of the Diatom Episome Maintenance Sequence and Improvement of Conjugation-Based DNA Delivery Methods.

Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here, we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in Escherichia coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in Phaeodactylum tricornutum. While various combinations of these individual elements enable efficient conjugation and high exconjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3′ end of HIS3 produced the highest number of diatom exconjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a high-throughput option utilizing 12-well plates and plating methods that improve exconjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance and demonstrate options for flexible vector design.

Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines.

Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems.

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I–VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.

PBR322 vectors having tetracycline-dependent replication

pBR322 vectors having tetracycline-dependent replication

Fast detection of deletion breakpoints using quantitative PCR.

The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories.