Genetic Engineering Approaches Research Articles

Recombinant Hybrid Proteins as Biospecific Reporters for Bioluminescent Microassay

Light-emitting proteins, luciferases and photoproteins, are widely used as reporters in high sensitive binding assays in vitro and in vivo. The combination of luciferase and biospecific molecule with affinity to a certain target (immunoglobulin, oligonucleotide, hapten, etc.) promotes specificity of the assay. The molecules of the kind (biospecific bioluminescent labels) are usually obtained by chemical synthesis using different commercial reagents. However, this approach cannot provide homogeneity of the product; it causes the loss (sometimes essential) of specific activity of the initial molecules. As a rule chemical binding requires a thorough verification of the reaction conditions (balance of reagents, pH and temperature of the media, reaction duration, etc.) that would decrease a negative impact of the reagents and improve the yield of conjugates. Another way to obtain combination of the kind is the creation of a hybrid protein by using genetic engineering approach. In this case a genetic construction containing, in one frame, genes coding biospecific and reporter proteins as well as sequences, coding a linker between them and sometimes auxiliary polypeptides, is created. Gene fusion approach provides easy bacterial production, hybrids homogeneity, easy purification using auxiliary polypeptides etc. The paper describes several hybrids “luciferase–biospecific polypeptide” designed in the photobiology lab of the Institute of Biophysics SB RAS, their construction, production and application in microassay as biospecific bioluminescent probes

The progress of genetic improvement in alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) is a perennial and outcrossing species, widely grown as a forage legume for hay, pasture and silage. The genetic engineering approaches involve the transfer of useful or novel gene(s) into alfalfa to improve desired traits. The recent development of genetic engineering is extensively applied to basic and applied research for alfalfa improvement, including improvement of herbicide resistance, reinforcement of the resistance to biotic and abiotic stresses, production of novel compounds, improved yield for industrial and/or pharmaceutical proteins and renewable energy sources. On the other hand, alfalfa forage needs to possess additional fermentable carbohydrates, proteins with a balanced amino acid profile that are gradually degraded in the rumen of domestic livestock, and zero anti-nutritional factors, which are the major concerns of recent interest in alfalfa. However, an advance of transgenic approach is contentious, requiring vigilant experimental methods and design to contest bio-safety challenges. More importantly, the technology of clustered regularly interspaced short palindromic repeats (CRISPR) is rapidly growing and might be a game player or changer in alfalfa. The present review can enable us to identify the proper direction, get familiar with new research methods and success of genetic engineering technology in alfalfa, to produce maximally improved cultivars.

An inducible expression system in the alga Nannochloropsis gaditana controlled by the nitrate reductase promoter

The marine microalgal species Nannochloropsis gaditana has emerged as a model organism for research into algal-derived biofuels due to its ability to produce large amounts of lipids. In addition, this species has biotechnological potential owing to its ease of transformation and availability of a sequenced and annotated genome. The ability to control expression of transgenes in an inducible manner is an important requirement for many genetic engineering approaches and as such, inducible promoters are an important component of the molecular toolkit for a host organism. We designed an expression vector containing a ~ 1.3 kb region upstream of the endogenous nitrate reductase gene of N. gaditana and demonstrated this is capable of promoting the expression of a heterologous enhanced green fluorescent protein (eGFP) reporter. In the presence of ammonium, expression of the eGFP reporter was undetectable; however, in the presence of nitrate, eGFP expression is induced. In addition, we demonstrated how altering the nitrogen source of the growth media can be used to precisely control expression. We have shown that the nitrate reductase promoter can be used as a powerful molecular tool for heterologous protein expression in N. gaditana, further contributing to the development of this species as a candidate strain for biotechnology.

Basic Engineering Strategies for Virus-Resistant Plants

Viral diseases of plants remain the main causes of significant damage in agricultural production. Various epidemiological measures have been taken to avoid viral diseases, including phytosanitary measures, using virus-free planting material, the fight against viral vectors, and the introduction of resistant or tolerant varieties. In recent years, the efforts of researchers around the world have been aimed at investigating the factors related to both natural and induced virus resistance of plants as well as the development and introduction of varieties created by genetic manipulation (bioengineering methods). The development of new antivirus strategies requires deep understanding of the molecular mechanisms underlying the natural resistance and biochemical processes occurring in virus infected plants. The review provides a brief description of genetic modification methods and considers the main genetic engineering approaches used to develop virus-resistant plants.

Genome and transcriptome of the natural isopropanol producer Clostridium beijerinckii DSM6423

BackgroundThere is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol.ResultsThe complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol.ConclusionsWe present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches.

Growing more with less: Breeding and developing drought resilient soybean to improve food security

Abstract Soybean (Glycine max L.) is considered an important crop, and enhancing its production under drought stress and changing climatic conditions to meet the challenges of global food security is crucial. Drought is considered one of the major abiotic factors that adversely affect soybean production. To address this challenge, we need to develop an integrative research approach that will bring together computational modelling, soil engineering, physiology, microbiology, molecular biology, genomics and plant breeding to study the underlying mechanisms of plant tolerance to drought stress. Advances in genomic technologies coupled with breeding approaches have helped scientists unravel the genes responsible for drought tolerance in crops. The success of a soybean breeding programme is largely dependent upon the extent of genetic variation in terms of drought tolerance-related traits. The availability of the whole soybean genome sequence is considered a major landmark in this process that will help design future strategies for improving soybean production. This review describes the recent advances in the study of the effect of drought stress on morpho-biochemical changes in soybean and the application of plant growth-promoting rhizobacteria, marker assisted selection, and different omics approaches to unravel the mechanism of drought tolerance. Here, in this review, we also highlight the customary knowledge on physiology, functional genomics and molecular breeding that may be significant in integrating genetic engineering and breeding approaches to improve drought tolerance in soybean crop.

Orchid floral volatiles: Biosynthesis genes and transcriptional regulations

Abstract Floral scent and colour are key trait for many floricultural crops. Floral volatiles are biologically and economically important plant derived compounds and they play vital role in pollinator attraction, plant defense and interaction with surrounding the environment. Orchidaceae is one of the largest and most widespread families of flowering plants, with more than 25,000 species. Orchids are one of the most significant plants in ecologically adopted every habitation on earth. Floral volatiles is not limited in its role to pollinator attraction, it’s widely used for perfumes, cosmetics, flavourings and therapeutic applications. Even though biochemistry of floral volatiles is still new field for investigation.Due to importance of the plant biology orchid floral scent research were deeply needed. Analysis of the biosynthesis mechanisms involved in floral scent is necessary to understand the fine-scale molecular functions and to breed to new cultivars through floral volatile genetic engineering approaches. Now a days many researchers developed floral scent models in plants. However, little is known about the pathways responsible for floral scent in orchids, due to the long life cycle and large genome size. Only some terpenoid pathways were reported in orchids. In this review, we mainly concentrated orchid floral volatile regulation and compounds synthesis responsible pathways. Moreover,we mentioned emission of orchid floral volatiles and their function in pollination ecology. This information will provide a basic information on orchid floral scent research.

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice.

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.

Transcriptomic and Functional Analyses Reveal That PpGLK1 Regulates Chloroplast Development in Peach (Prunus persica).

Peach is an ideal species for fruit tree research because of its small, fully sequenced genome. Chloroplast development is dependent on the tight cooperation between the nuclear and plastid genomes, and is regulated by GLK transcription factors. In this work, the pigment content was monitored and the chloroplast-to-chromoplast conversion during the fruit ripening was visualized by transmission electron microscopy. Localization and expression analyses showed that PpGLK1 was located in the nucleus and expressed mainly in the leaves and fruit skin. A transcriptome analysis showed that PpGLK1 and its target genes were significantly differentially expressed in ripening peach fruit skin. PpGLK1 silencing affected chlorophyll accumulation in peach leaves and fruits. Overexpression of PpGLK1 rescued the phenotypes of the Arabidopsis Atglk1Atglk2 double mutant and the tomato uniform ripening mutant. The results of a yeast two-hybrid analysis showed that PpGLK1 is autoactivated and that PpGLK1 (301-542 a.a.) interacted with PpARF5. Together, our results indicate that PpGLK1 regulates chloroplast development in green tissues in peach. Therefore, it may be a promising target gene for improving the production and quality of peach by genetic engineering and breeding approaches.

Ocular delivery of proteins and peptides: Challenges and novel formulation approaches.

The impact of proteins and peptides on the treatment of various conditions including ocular diseases over the past few decades has been advanced by substantial breakthroughs in structural biochemistry, genetic engineering, formulation and delivery approaches. Formulation and delivery of proteins and peptides, such as monoclonal antibodies, aptamers, recombinant proteins and peptides to ocular tissues poses significant challenges owing to their large size, poor permeation and susceptibility to degradation. A wide range of advanced drug delivery systems including polymeric controlled release systems, cell-based delivery and nanowafers are being exploited to overcome the challenges of frequent administration to ocular tissues. The next generation systems integrated with new delivery technologies are anticipated to generate improved efficacy and safety through the expansion of the therapeutic target space. This review will highlight recent advances in formulation and delivery strategies of protein and peptide based biopharmaceuticals. We will also describe the current state of proteins and peptides based ocular therapy and future therapeutic opportunities.

Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy.

The remarkable progress in engineering and clinical development of therapeutic antibodies in the last 40 years, after the seminal work by Köhler and Milstein, has led to the approval by the United States Food and Drug Administration (FDA) of 21 antibodies for cancer immunotherapy. We review here these approved antibodies, with emphasis on the methods used for their discovery, engineering, and optimization for therapeutic settings. These methods include antibody engineering via chimerization and humanization of non-human antibodies, as well as selection and further optimization of fully human antibodies isolated from human antibody phage-displayed libraries and immunization of transgenic mice capable of generating human antibodies. These technology platforms have progressively led to the development of therapeutic antibodies with higher human content and, thus, less immunogenicity. We also discuss the genetic engineering approaches that have allowed isotype switching and Fc modifications to modulate effector functions and bioavailability (half-life), which together with the technologies for engineering the Fv fragment, have been pivotal in generating more efficacious and better tolerated therapeutic antibodies to treat cancer.

Analysis of Insulin Analogs and the Strategy of Their Further Development.

We analyzed the structural properties of the peptide hormone insulin and described the mechanism of its physiological action, as well as effects of insulin in type 1 and 2 diabetes. Recently published data on the development of novel insulin preparations based on combining molecular design and genetic engineering approaches are presented. New strategies for creation of long-acting insulin analogs, the mechanisms of functioning of these analogs and their structure are discussed. Side effects of insulin preparations are described, including amyloidogenesis and possible mitogenic effect. The pathways for development of novel insulin analogs are outlined with regard to the current requirements for therapeutic preparations due to the wider occurrence of diabetes of both types.

Quantification of Photosynthetic Enzymes in Leaf Extracts by Immunoblotting.

In this chapter, we describe a method to extract and quantify photosynthetic enzymes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The method is particularly suitable for characterizing altered protein amounts in leaves of plants produced from genetic engineering or gene-editing approaches. We focus on RuBisCO and RuBisCO activase, a molecular chaperone required to sustain the activity of RuBisCO and CO2 fixation, yet the method can be easily adapted to investigate other leaf proteins of interest.

Zebrafish: A Pharmacogenetic Model for Anesthesia.

General anesthetics are small molecules that interact with and effect the function of many different proteins to promote loss of consciousness, amnesia, and sometimes, analgesia. Owing to the complexity of this state transition and the transient nature of these drug/protein interactions, anesthetics can be difficult to study. The zebrafish is an emerging model for the discovery of both new genes required for the response to and side effects of anesthesia. Here we discuss the tools available to manipulate the zebrafish genome, including both genetic screens and genome engineering approaches. Additionally, there are various robust behavior assays available to study anesthetic and other drug responses. These assays are available for single-gene study or high throughput for genetic or drug discovery. Finally, we present a case study of using propofol as an anesthetic in the zebrafish. These techniques and protocols make the zebrafish a powerful model to study anesthetic mechanisms and drug discovery.

In Planta Production of Fluorescent Filamentous Plant Virus-Based Nanoparticles.

Viral nanoparticles are attractive platforms for biomedical applications and are frequently employed for optical imaging in tissue culture and preclinical animal models as fluorescent probes. Chemical modification with organic dyes remains the most common strategy to develop such fluorescent probes. Here we report a genetic engineering approach to incorporate fluorescent proteins in viral nanoparticles, which can be propagated in their plant host. The fluorescent viral nanoparticles so obtained obviate post-harvest modifications and thereby maximize yields. Our engineering approach transforms filamentous potato virus X (PVX) to display green fluorescent protein (GFP) or mCherry as N-terminal coat protein (CP) fusions at a 1:3 fusion protein to CP ratio through integration of the foot-and-mouth disease 2A sequence. The in planta propagation of recombinant GFP-PVX or mCherry-PVX thus produced in Nicotiana benthamiana can be easily documented using fluorescence imaging. Molecular farming protocols can be accordingly optimized by monitoring chimera stability over the course of the infection cycle. Moreover, we also demonstrate the utility of recombinant mCherry-PVX in optical imaging of human cancer cells and tumor tissue in preclinical mice model. Together, these features make genetically engineered fluorescent PVX particles ideally suited for molecular imaging applications.

CRISPR-Cas9: a promising genetic engineering approach in cancer research.

Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions in in vitro and in vivo environments. The accuracy and versatility of CRISPR-Cas have been capitalized upon in biological and medical research and bring new hope to cancer research. Cancer involves complex alterations and multiple mutations, translocations and chromosomal losses and gains. The ability to identify and correct such mutations is an important goal in cancer treatment. In the context of this complex cancer genomic landscape, there is a need for a simple and flexible genetic tool that can easily identify functional cancer driver genes within a comparatively short time. The CRISPR-Cas system shows promising potential for modeling, repairing and correcting genetic events in different types of cancer. This article reviews the concept of CRISPR-Cas, its application and related advantages in oncology.

Informed consent in field trials of gene-drive mosquitoes.

The US National Academies' (NAS) recent report 'Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values' examines the requirements of responsible conduct in research involving gene drives in non-human organisms. Many of the complex ethical issues raised by the introduction of gene drive technologies for mosquito population control have been anticipated during the development and field-testing of earlier-generation genetic engineering approaches with mosquitoes. One issue-the requirement for informed consent in field trials-is not addressed explicitly in the NAS' report. Some commentators have presumed that informed consent should play a role as a protection for research participants in studies of genetically modified mosquitoes. Others have argued that there are no human subjects of field trials, so the informed consent requirement does not apply. It is both ethically and practically important that these presumptions are adequately scrutinized to ensure that any applications of informed consent in these trials are properly justified. We argue that informed consent from individual research participants in gene drive trials may be required: (1) when blood and other forms of clinical data are collected from them, as will likely be the case in some studies involving epidemiological endpoints, such as the incidence of new infections with dengue and malaria; (2) when they participate in social science and/or behavioral research involving the completion of surveys and questionnaires; or (3) when their home or property is accessed and the location recorded as a spatial variable for the release or collection of mosquitoes because the precise location of the household is important for entomological reasons and these data constitute identifiable private information at the household level. Importantly, most regulations and guidelines allow these requirements to be waived or modified, to various degrees, according to the judgment of Institutional Review Boards.

Virus resistance in potato: Current state and prospects

The potato (Solanum tuberosum), one of the world’s most important food crops, is infected by numerous viruses, nine of which are of major economic importance as they cause significant yield losses and reduce the quality of the crop. To mitigate the consequences of viral infections, countries with highly developed agriculture are improving and promoting sanitary measures, which include constantly monitoring viruses and certification of potato seed material based on virus diagnostics and improved potato cultivars. However, the development of virus-resistant potato cultivars seems to be a preferable choice in the long term. By employing conventional breeding techniques and genetic engineering approaches using natural virus resistance genes, normally obtained from the wild growing Solanum species, or virus-specific sequences, a number of potato cultivars/lines, which are resistant to the majority of known potato viruses, have already been obtained. However, the above-mentioned techniques have certain limitations, which are determined, in particular, by the high virus specificity of the acquired resistance (against certain viruses only), its short duration, and the ability of the virus to overcome it, as well as the regularly declared bans on the use of genetically modified plants. In the modern era, the novel technologies allowing us to edit genomes for the purposes of gene design have created the possibility to engineer a new generation of resistance genes. The most promising approaches are thought to be the directed mutagenesis of resistance genes, which allows broadening the spectrum of the effects of the gene and the use of nonhost resistance (NHR) (nonspecific resistance) genes, allowing engineering plants resistant to unrelated viruses and in certain cases, to other pathogens and even abiotic stresses. The identification of genes involved in the mechanisms of NHR has only just begun. The nucleus is now being considered as a new source of still unknown factors involved in diverse signal pathways controlling the plant’s defensive response against virus infection. The review describes the approaches which are used to generate virus-resistant potato plants and the challenges faced in this path.

Short communication: Genomic and phenotypic analyses of exopolysaccharides produced by Streptococcus thermophilus KLDS SM

Streptococcus thermophilus plays important roles in the dairy industry. Streptococcus thermophilus KLDS SM could produce a high amount of exopolysaccharides (EPS). To understand the possible link between the genotype and the phenotype regarding EPS, the complete genome of S. thermophilus KLDS SM was sequenced and investigated in silico for genes related to carbohydrate fermentation, nucleotide sugars synthesis, and EPS gene cluster. We found that S. thermophilus KLDS SM is able to ferment sucrose, mannose, glucose, galactose, and lactose from the genomic research, which was confirmed by API 50 CH (bioMérieux, Marcy l'Etoile, France). The genetic analysis of nucleotide sugars and EPS cluster revealed that the EPS produced by this strain are composed of galactose and glucose, in accordance with the biochemical result. Furthermore, differences in the molecular mass of EPS from S. thermophilus KLDS SM cultivated under different carbon sources were correlated with the transcription levels of the genes encoding chain length determination protein and glycosyltransferase. Our findings provide a better understanding of the link between the genetic elements and the chemical conformation of EPS and a theoretical basis for producing tailor-made EPS through genetic and metabolic engineering approaches.

Nanoscale wide-band semiconductors for photocatalytic remediation of aquatic pollution.

Water pollution is a serious challenge to the public health. Among different forms of aquatic pollutants, chemical and biological agents create paramount threat to water quality when the safety standards are surpassed. There are many conventional remediatory strategies that are practiced such as resin-based exchanger and activated charcoal/carbon andreverse osmosis. Newer technologies using plants, microorganisms, genetic engineering, and enzyme-based approaches are also proposed for aquatic pollution management. However, the conventional technologies have shown impending inadequacies. On the other hand, new bio-based techniques have failed to exhibit reproducibility, wide specificity, and fidelity in field conditions. Hence, to solve these shortcomings, nanotechnology ushered a ray of hope by applying nanoscale zinc oxide (ZnO), titanium dioxide (TiO2), and tungsten oxide (WO3) particles for the remediation of water pollution. These nanophotocatalysts are active, cost-effective, quicker in action, and can be implemented at a larger scale. These nanoparticles are climate-independent, assist in complete mineralization of pollutants, and can act non-specifically against chemically and biologically based aquatic pollutants. Photocatalysis for environmental remediation depends on the availability of solar light. The mechanism of photocatalysis involves the formation of electron-hole pairs upon light irradiations at intensities higher than their band gap energies. In the present review, different methods of synthesis of nanoscale ZnO, TiO2, and WO3 as well as their structural characterizations have been discussed. Photodegradation of organic pollutants through mentioned nanoparticles has been reviewed with recent advancements. Enhancing the efficacy of photocatalysis through doping of TiO2 and ZnO nanoparticles with non-metals, metals, and metal ions has also been documented in this report.