Gibson Assembly Research Articles

Novel trimethoprim resistance gene, dfrA35, in IncC plasmids from Australia.

In Gram-negative bacteria, over 30 different genes are known to encode a trimethoprim-insensitive dihydrofolate reductase that confers resistance to trimethoprim. To determine whether a gene encoding a putative dihydrofolate reductase found in type 2 IncC plasmids isolated between 2002 and 2013 in healthcare facilities in Melbourne, Australia, confers trimethoprim resistance. Conjugation was used to transfer plasmids into a laboratory Escherichia coli. A PCR-amplified fragment was cloned into pUC19 using Gibson Assembly and transformed into E. coli. The level of resistance to trimethoprim was determined using broth microdilution. MEGA (7.0.26) and Geneious Prime (7.0.9) were used to examine the relationship to known Dfr proteins. The conjugative IncC plasmid pEc158 from a 2002 Melbourne clinical E. coli isolate was shown to transfer trimethoprim resistance. The putative DfrA protein encoded by a dfrA gene in pEc158 shares <40% amino acid identity with any previously identified DfrA protein. This gene was cloned and found to confer trimethoprim resistance. The gene and protein were named dfrA35/DfrA35. In pEc158 the dfrA35 gene is located near the ori end of a partial copy of the CR1 element, within a complex resistance island. It is found in the same location in further closely-related type 2 IncC plasmids from Klebsiella pneumoniae (Melbourne, 2013), which were not transfer proficient. Resistance determinants continue to be found and will be missed using website-associated databases to infer phenotypes from genome sequences rather than direct phenotypic testing.

FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E-cadherin and CD44 expression.

Circular RNAs (circRNAs) are a new class of RNAs, and many studies have identified thousands of circRNAs in tumor cells. Fibronectin type III domain‐containing protein 3B (FNDC3B) circular RNA (circFNDC3B, circBase ID: hsa_circ_0006156) circularizes with exons 5 and 6. Gibson Assembly DNA technology was used to construct a circFNDC3B expression vector without a splice site and restriction enzyme site. We showed that circFNDC3B increased migration and invasion in gastric cancer (GC). Ectopic expression of circFNDC3B reduced the level of E‐cadherin protein to promote the epithelial–mesenchymal transition in GC. RNA immunoprecipitation assays and RNA pull‐down assays confirmed that circFNC3B increased CD44 expression, which was associated with cell adhesion, via the formation of a ternary complex of circFNDC3B‐IGF2BP3‐CD44 mRNA. These results indicated that circFNDC3B was associated with the degree of malignancy to highlight the specific characteristics of cell invasion.

Molecular Cloning of a Fluorescence‐Based Reporter Plasmid for Cell‐Free Protein Synthesis

Cell‐free protein expression, also known as in vitro protein synthesis is a convenient and fast method for protein production. It bypasses the need to culture living cells, which can be time and resource consuming. Among multiple cell‐free protein expression systems available, the wheat germ extract system has become a popular choice for producing eukaryotic proteins due to high yields and commercial availability. The goal of this work was to generate a fluorescence‐based reporter plasmid to be used in wheat germ based cell‐free protein expression system. Because of its unique optical properties, the Green Fluorescent Protein (deGFP) can be used as a positive control during high‐throughput synthesis of proteins by a cell‐free system. To construct the reporter plasmid, Gibson Assembly strategy was used to clone a deGFP gene in a wheat germ extract compatible plasmid. The deGFP gene was successfully cloned in two plasmids, bearing a hexahistidine tag on either the N‐, or C‐terminus of GFP. The clones were validated by Sanger DNA sequencing. After validation, protein expression was confirmed by western blotting using an anti‐His antibody. The reporter plasmid will be used as a positive control during high‐throughput production of eukaryotic transcription factors by wheat germ cell free protein synthesis.Support or Funding InformationResearch Initiative for Scientific Enhancement (RISE), GRANT 5R25GM061151‐17This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Generation of barcoded plasmid libraries for massively parallel analysis of chromatin position effects

The discovery of the position effect variegation phenomenon and the subsequent comprehensive analysis of its molecular mechanisms led to understanding that the local chromatin composition has a dramatic effect on gene activity. To study this effect in a high-throughput mode and at the genome-wide level, the Thousands of Reporters Integrated in Parallel (TRIP) approach based on the usage of barcoded reporter gene constructs was recently developed. Here we describe the construction and quality checks of high-diversity barcoded plasmid libraries supposed to be used for high-throughput analysis of chromatin position effects in Drosophila cells. First, we highlight the critical parameters that should be considered in the generation of barcoded plasmid libraries and introduce a simple method to assess the diversity of random sequences (barcodes) of synthetic oligonucleotides using PCR amplification followed by Sanger sequencing. Second, we compare the conventional restriction-ligation method with the Gibson assembly approach for cloning barcodes into the same plasmid vector. Third, we provide optimized parameters for the construction of barcoded plasmid libraries, such as the vector : insert ratio in the Gibson assembly reaction and the voltage used for electroporation of bacterial cells with ligation products. We also compare different approaches to check the quality of barcoded plasmid libraries. Finally, we briefly describe alternative approaches that can be used for the generation of such libraries. Importantly, all improvements and modifications of the techniques described here can be applied to a wide range of experiments involving barcoded plasmid libraries.

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems.

Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes. In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9

Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.

Developing a universal strategy for cloning and assembly of the genomes of diverse Epstein – Barr virus strains

Epstein-Barr virus (EBV), is an oncogenic gamma-herpesvirus, which is associated with malignant diseases of B cells, T cells, and epithelial cells. EB viruses have large DNA genomes of more than 170 kb that are difficult to clone and manipulate. Here we describe 2 different approaches for cloning whole EBV genomes of diverse strains for reverse genetics studies. The first approach used CRISPR/Cas9-mediated cloning of the entire EBV genome into a bacterial artificial chromosome (BAC) vector using homologous recombination in B cells. This method allowed the cloning of the type 2 EBV strain Jijoye for the first time, but the BAC-clones are unstable. This strategy is being modified by recoding the homology regions to make the clones more stable. The second approach involves transformation-associated recombination (TAR) cloning of EBV fragments and their assembly in yeast, which will allow for mixing and matching DNA regions from different EBV strains for functional studies. This approach is based on TAR cloning of the EBV genome as 10 overlapping fragments, which average 17 kilobases long, using the natural homologous recombination processes of the yeast. Subsequent assembly of all the overlapping fragments is undertaken in yeast or by Gibson assembly to reconstitute the infectious EBV clone. Two fragments from EBV strains B95-8 and AG876 were captured and isolated successfully, but at low efficiency. We are currently improving the TAR cloning efficiency by increasing the size of the capture homology regions to approximately 500 bp coupled with CRISPR/Cas-9-mediated fragmentation of the EBV genome.

The Removal of Histones from Ultrafine Anaphase Bridges Studied by Optical Tweezers

During mitosis, duplicated DNA has to be faithfully segregated into two daughter cells. In anaphase, regions of entangled double-stranded DNA between segregating sister chromatids can give rise to DNA threads known as ultrafine anaphase bridges (UFBs). Failure to resolve UFBs can result in genome instabilities. It has been proposed that a key step in UFB resolution is the removal of histones, and that this is facilitated by the SNF2-family protein PICH. The mechanism of nucleosome remodeling by PICH has, however, remained elusive. Here, we present a single-molecule assay that recapitulates chromatin re-organization during UFB resolution. In vitro chromatin constructs consisting of 4 to 12 high affinity histone binding sites (601 sequences) within an 8.5 kbp DNA molecule were prepared using a combination of restriction-enzyme based cloning and Gibson assembly. After reconstitution with human histones, these constructs were manipulated within a multi-channel microfluidic flow cell using dual-trap optical tweezers. This approach allows us to measure tension-induced nucleosome unwrapping in the presence or absence of PICH. Our results suggest that, under moderate tension (10 pN), PICH facilitates the unwrapping of nucleosomes. In further experiments, we aim to correlate nucleosome stability with the dynamics of single fluorescently-labelled PICH proteins on DNA. Taken together, our single-molecule approach provides new insights into the mechanisms by which PICH is involved in anaphase UFB resolution.

The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin

Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an iron-regulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2-/- mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.

Reconstruction and validation of three different binary vectors suitable for generation of genetically engineered Helicoverpa protected crops

Availability of a suitable plant transformation binary vector is necessary for the generation of transgenic crops with an adequate expression of transgenic proteins. Therefore, three binary vectors were constructed viz., pBK204, pBK205, and pBK206 harboring either a truncated or a full-length version of a Cry1Ac gene for the generation of Helicoverpa protected crops. Two different promoters viz., Arabidopsis Rubisco small subunit (AtSSU) gene promoter or CaMV35S promoters were used to regulate the various versions of Cry1Ac gene. The binary vectors were reconstructed either by the Gibson assembly method and others by ligating the restriction enzyme digested fragments. The reconstructed binary vectors were mobilized into Agrobacterium strain AGL1 and validated by Agrobacterium infiltration assays of Nicotiana benthamiana. The amount of Cry1Ac protein accumulated in the Agroinfiltrated tobacco leaves was quantified using the quantitative ELISA assay. The expression of the Cry1Ac protein in the tobacco leaves ranged from 0.25 to 0.26 µg /g fresh weight (FW) when transformed with these three constructs. Thus, the vectors constructed in this study appeared to be suitable for generation of Helicoverpa resistant transgenic crops by Agrobacterium-mediated genetic transformation method.

Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain.

Recently developed gene editing technologies based on engineered CRISPR/Cas9 systems enables researchers to disrupt genes in a cell type-specific manner in the adult mouse brain. Using these technologies, we recently showed that the dopamine beta-hydroxylase gene in Locus Coeruleus (LC) norepinephrine neurons plays a vital role in the maintenance of wakefulness. Our method consists of four steps, (1) crossing Cre-dependent spCas9 knockin mice with a Cre-driver mouse line to express spCas9 in the target neural populations, (2) cloning of sgRNA, (3) construction of an AAV (adeno associated virus) vector expressing dual sgRNA, and (4) virus packaging and stereotaxic injection of the virus into the target brain area. Here, we describe a detailed protocol of AAV vector construction for cell type-specific CRISPR gene editing in the adult mouse brain. The method adopts a dual-sgRNA strategy for efficient disruption of the target gene. At first, a few different sgRNAs targeting the same gene are cloned into a plasmid expressing spCas9. After evaluation of the sgRNAs by a T7 endonuclease assay, the two most efficient sgRNAs are cloned in tandem into an AAV vector using the Gibson Assembly method.

SmartBac, a new baculovirus system for large protein complex production

Recent revolution of cryo-electron microscopy has opened a new door to solve high-resolution structures of macromolecule complexes without crystallization while how to efficiently obtain homogenous macromolecule complex sample is therefore becoming a bottleneck. Here we report SmartBac, an easy and versatile system for constructing large-sized transfer plasmids used to generate recombinant baculoviruses that express large multiprotein complexes in insect cells. The SmartBac system integrates the univector plasmid-fusion system, Gibson assembly method and polyprotein strategy to construct the final transfer plasmid. The fluorescent proteins are designed co-expressed with the target to monitor transfection and expression efficiencies. A scheme of screening an optimal tagged subunit for efficient purification is provided. Six large multiprotein complexes including the human exocyst complex and dynactin complex were successfully expressed and purified, suggesting a great potential of SmartBac system for its wide application in the future.

Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging

Endothelial cells of the vascular system are dynamic cells whose molecular adaptability is decisive for the adjustment of homeostasis and organ perfusion. Advanced microscopic techniques, automation processing, and image analysis software was shown to improve the understanding of vascular biology. In this work, we describe advanced methods that allow investigating the dynamics of endothelial cell contacts. The development of viral vectors has contributed significantly to the genetic manipulation of endothelial cells. We used the Gibson assembly as a quick and cheap cloning system for introducing sequences into the lentiviral-based pFUGW vector. Furthermore, classical fluorescence tags such as mCherry and EGFP were compared with self-labeling tags such as Halo and SNAP for their suitability to study junction dynamics in cultured endothelium, and found the self-labeling tags as useful tools. Using such combinations, we found maintained cell junction integrity during shear stress-induced junction remodeling using VE-cadherin-EGFP. Remodeling was accompanied by VE-cadherin plaque formation, indicating that this process is mediated by the for­mation of the actin-driven junction-associated intermittent lamellipodia, JAIL. The combined methods including the Gibson assembly, lentiviral mediated gene transfer, spinning disk-based live cell imaging, and software for quantification allow analyses of the endothelial cell junction dynamics under static and under shear stress conditions.

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus.

SummaryFungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.

AMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes

Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of “multiple integrases-multiple attB sites”. This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts.

Role of AML1-ETO Related Fusion Circular RNA in the Growth of Leukemia Cells

Introduction: The pathogenesis of acute myeloid leukemia (AML) is always associated with chromosomal translocation, such as t(8;21)(q21;q22), which results in the formation of AML1-ETOfusion gene. The AML1-ETO fusion protein leads to hematopoietic differentiation blocked and leukemogenesis. How genomic abnormities such as chromosomal translocation influence the production and biological function of noncoding RNAs is relatively less studied. With the application of high-throughput sequencing and bioinformatics, it is increasing clear that the circular RNAs (circRNA) played important role in multiple biological processes. However, circRNA is rarely studied in hematopoietic malignancies. Here we assessed the roles of AML1-ETO related fusion circRNA (F-CircAE) in AML1-ETO leukemia.Methods: Total RNA was extracted from leukemia cell lines Kasumi-1 and SKNO-1, then treated with RNaseR or not. PCR assay with divergent primers was performed to identify F-CircAEs. The F-CircAE retroviral expression vector was constructed using Gibson Assembly Cloning Kit. NIH3T3 cells were transduced with retrovirus, the proliferation and foci formation were detected by crystal violet staining. F-CircAE-expressingNIH3T3 cells were injected subcutaneously into nude mice. Tumor growth was monitored at indicated time. To knock down the expression of F-CircAEs, the short hairpin double-stranded oligo targeting the back-splice junction of the F-CircAEs was inserted into the pLKO.1 lentiviral vector. Kasumi-1 cells were transduced with lentivirus, then the proliferation and cell cycle distribution of Kasumi-1 cells was measured by MTS assay and PI staining. Western blot was used to detect the cell cycle associated protein expression. RNA pulldown assay was performed with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the instructions. The bound proteins in the pulldown assay were analyzed by spectrum analysis. RNA-seq were performed using the Illumina platform. Sequencing reads were analyzed by a standard RNA-Seq pipeline. Gene Set Enrichment Analysis (GSEA) were used to analyze the pathway enrichment.Results: Several F-CircAEswere identified in AML1-ETO leukemia cell lines (Kasumi-1, SKNO-1) and t(8;21) patients' bone marrow mononuclear cells (BMMCs), but not in K562 cells and donors' BMMCs (Figure 1). To further validate F-CircAEs, RNaseR treatment was conducted and amplification products of circular RNAs were still detectable in AML1-ETO positive cells (Figure 1). Subsequently, Sanger sequencing of these amplification circular products revealed the head to tail sites of F-CircAE.To examine the function of F-CircAE, overexprseeion of F-CircAE in NIH3T3 cells resulted in increased proliferation compared with mutated F-CircAE (F-CircAE-mut) overexprssion or control group (Figure 2). Meanwhile, more foci were formed in the overexpressed F-CircAE group. In vivo,F-CircAE overexpression also promoted the growth of NIH3T3 cells in the injection site of nude mice. Furthermore, silencing of two F-CircAEs reduced the growth rates of Kasumi-1 cells (Figure 3) and induced an increase in G1 phase and a decrease in S and G2/M phase compared with scramble group. The protein levels of CDK2 and CyclinD1, which were the cell cycle regulatory components at G1 boundary, were reduced in F-CircAE knockdown groups compared with scramble group. To explore the mechanism by which F-CircAE promoted leukemia cells proliferation, RNA-pulldown assay was performed and more than 70 proteins were found to bind to F-CircAE. Based on the sequencing reads of RNA-seq, GSEA revealed several pathways were enriched following F-CircAE knockdown in Kasumi-1 cells.Conclusions: F-CircAEs were existed in t (8;21) leukemia cell lines and primary AML leukemia patients' BMMCs. F-CircAE could bind to proteins involved in the growth of leukemia cells. Discovery of F-CircAEs in AML1-ETO leukemia could make an immense progress in understanding their pathogenic mechanism, indicating new marker for diagnosis and therapy target. DisclosuresNo relevant conflicts of interest to declare.

New approach for the construction of infectious clones of a circular DNA plant virus using Gibson Assembly

Viruses belonging to the genus Begomovirus (family Geminiviridae) have circular single-strand DNA genomes encapsidated into quasi-icosahedral particles, and are transmitted by whiteflies of the Bemisia tabaci complex. Biological and molecular properties of begomoviruses have been studied efficiently with infectious clones containing dimeric genomic components. However, current approaches employing enzymatic digestion and ligation to binary vectors are laborious, mostly due to many cloning steps or partial digestion by restriction enzyme. Here, an infectious clone of the bipartite begomovirus Bean golden mosaic virus (BGMV) was obtained using PCR and Gibson Assembly (GA). Common bean (Phaseolus vulgaris) seedlings displayed severe yellow mosaic and stunt symptoms 15 days after agroinoculation with DNA-A and DNA-B of BGMV. The approach based on PCR-GA protocol is a fast and useful tool to obtain infectious clones of a circular DNA plant virus.

Production of a Beet chlorosis virus full-length cDNA clone by means of Gibson assembly and analysis of biological properties.

Beet chlorosis virus (genus Polerovirus, family Luteoviridae), which is persistently transmitted by the aphid Myzus persicae, is part of virus yellows in sugar beet and causes interveinal yellowing as well as significant yield loss in Beta vulgaris. To allow reverse genetic studies and replace vector transmission, an infectious cDNA clone under cauliflower mosaic virus 35S control in a binary vector for agrobacterium-mediated infection was constructed using Gibson assembly. Following agroinoculation, the BChV full-length clone was able to induce a systemic infection of the cultivated B. vulgaris. The engineered virus was successfully aphid-transmitted when acquired from infected B. vulgaris and displayed the same host plant spectrum as wild-type virus. This new polerovirus infectious clone is a valuable tool to identify the viral determinants involved in host range and study BChV protein function, and can be used to screen sugar beet for BChV resistance.

Cellular reagents for diagnostics and synthetic biology.

We have found that the overproduction of enzymes in bacteria followed by their lyophilization leads to 'cellular reagents' that can be directly used to carry out numerous molecular biology reactions. We demonstrate the use of cellular reagents in a variety of molecular diagnostics, such as TaqMan qPCR with no diminution in sensitivity, and in synthetic biology cornerstones such as the Gibson assembly of DNA fragments, where new plasmids can be constructed solely based on adding cellular reagents. Cellular reagents have significantly reduced complexity and cost of production, storage and implementation, features that should facilitate accessibility and use in resource-poor conditions.

Generation and characterization of UL41 null pseudorabies virus variant in vitro and in vivo

BackgroundThe alphaherpesvirus virion host shutoff (vhs) gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis. The roles of vhs in HSV-1 and HSV-2 have been studied extensively in previous studies, however, relatively little is known about the vhs protein of PRV.MethodsA novel method combining CRISPR/Cas9 and Gibson assembly was developed to generate UL41 null PRV variant. The properties of UL41 null PRV in vitro and in vivo were further characterized. And the vhs activity of UL41 protein of PRV variant was evaluated by luciferase assay, Western-blot and RT-qPCR.ResultsGibson assembly based on homologous recombination can accomplish one-step insertion of viral DNA fragments into donor plasmids efficiently (> 80%). Cas9/gRNA further largely enhanced the efficiency of homologous recombination. Using this method we were able to rapidly generate the UL41 null and revertant viruses of PRV variant. Compared to wild type (JS-2012), the UL41 null virus showed significantly smaller plaques and lower titers in Vero cells and impaired lethality and neuroinvasion in mice. Further the UL41 protein from different PRV strains exhibited unequal vhs activity in vitro, which of JS-2012 showed significantly weaker vhs activity than that of European-American strains. In addition UL41 null virus can also significantly decrease the expression of host genes during the early period of infection, which suggests other viral factors may be also involved in host shutoff.ConclusionsCRISPR/Cas9 combined with Gibson assembly efficiently generated UL41 null PRV. Compared to wild type, UL41 null PRV showed impaired both replication capability in vitro and neuroinvasion in vivo. Further UL41 protein of PRV variant showed significantly weaker vhs activity than that of PRV SC (European-American-like strain), suggesting the deficiency of vhs activity by the PRV variant UL41 protein.