Use Of Vectors Research Articles

Fluid inclusion and multiple isotope geochemical constraints on the hydrothermal evolution and metal sources of the Qiaomaishan Cu–W skarn deposit, eastern China

The Xuancheng ore district of eastern China is a newly discovered district within the Middle and Lower Yangtze River Metallogenic Belt (MLYB) that hosts several magmato-hydrothermal related mineral deposits. The Qiaomaishan deposit is a representative example of the skarn deposits within this district and is also the only deposit within the district that hosts tungsten mineralization. However, the source of metals and the hydrothermal evolution of the mineralizing system that formed this deposit remain controversial. This study addresses these issues using new fluid inclusion microthermometric and isotopic data, including fluid inclusion H, quartz O, sulfide S, and pyrite Pb data, which constrain the evolution of the hydrothermal fluids within the system and the source of metal within the Qiaomaishan deposit. This study identified three main stages of hydrothermal evolution and paragenesis within the deposit, namely pre-ore (stage 1), syn-ore (stages 2 and 3), and post-ore formation stages. Stage 1 garnet (andradite)-hosted fluid inclusions homogenize between 390 °C and 590 °C and have salinities of 9–20 wt% NaCl equivalent whereas stage 2 quartz-hosted fluid inclusions homogenize between 200 °C and 460 °C and have salinities of 5–18 wt% NaCl equivalent. Finally, stage 3 quartz- and calcite-hosted fluid inclusions homogenize at temperatures of 120 °C–240 °C and 3–15 wt% NaCl equivalent salinities. The stage 2 quartz has oxygen and hydrogen isotopic compositions (δDfluid from −99 ‰ to −57 ‰; δ18Ofluid from 4.0 ‰ to 6.1 ‰) that are close to those expected for magmatic fluids, whereas stage 3 quartz (δDfluid from −105 ‰ to −86 ‰; δ18Ofluid from −1.7 to −0.6 ‰) records the mixing of meteoric and hydrothermal magmatic fluids. These fluid inclusion data suggest that cooling was the main mineralizing process involved in stages 1 and 2, and this process may favor the enrichment of tungsten and copper in the residual hydrothermal fluids. In contrast, fluid mixing became increasingly important between stages 2 and 3, leading to a reduction in salinity and temperature as well as changes in fluid isotopic compositions. Water–rock (W/R) interaction is also likely to have been an important process during deposit formation. The δ34S (2.7 ‰–5.7 ‰ with a mean of 4.3 ‰) of sulfides from the Qiaomaishan deposit also provide evidence that the sulfur within the deposit has a magmatic origin. The homogeneous pyrite Pb isotopic data (206/204Pb = 18.158–18.518, 207/204Pb = 15.592–15.650, and 208/204Pb = 36.179–38.634) for samples from the Qiaomaishan deposit further indicates that the metals within the deposit were derived from both mantle and crustal sources. In summary, the Qiaomaishan deposit formed from hydrothermal fluids derived from a magmatic source that subsequently cooled, mixed with meteoric water, and underwent W/R interaction, causing sulfide precipitation. The large-scale folding present in this area may also have been a key focus for mineralizing fluids and this a potential vector for use in mineral exploration as these structures focused both magmatism and fluid flow, promoting the formation of the skarn mineralization in this region.

New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae

The emergence of antibiotic-resistant Klebsiella pneumoniae is a significant global health threat that has led to increased morbidity and mortality. This resistance also hinders basic research, as many strains are no longer susceptible to antibiotics commonly used in microbial genetics. Addressing this requires the development of new genetic tools with alternative selective markers. In this report, we introduce new allelic exchange vectors for use in drug-resistant strains. These vectors feature a conditional R6K origin of replication, an origin of transfer, SacB counter-selection, and alternative selectable markers. We validated the vectors by generating unmarked deletions in the K. pneumoniae KPPR1S bla (β-lactamase) and lacZ (β-galactosidase) genes. During this process, we defined optimized conditions for SacB-mediated allelic exchange in KPPR1S, significantly enhancing the efficiency of mutant generation. Furthermore, we demonstrated that lacZ is dispensable for virulence and that the lacZ mutant can serve as a surrogate for wild-type strains in competition assays using the Galleria mellonella infection model. Our findings provide new tools for the efficient genetic manipulation of K. pneumoniae and other drug-resistant bacteria.

Improved Feedback Quantizer with Discrete Space Vector.

The use of advanced modulation and control schemes for power converters, such as a Feedback Quantizer and Predictive Control, is widely studied in the literature. This work focuses on improving the closed-loop modulation scheme called Feedback Quantizer, which is applied to a three-phase voltage source inverter. This scheme has the natural behavior of mitigating harmonics at low frequencies, which are detrimental to electrical equipment such as transformers. This modulation scheme also provides good tracking for the voltage reference at the fundamental frequency. On the other hand, the disadvantage of this scheme is that it has a variable switching frequency, creating a harmonic spectrum in frequency dispersion, and it also needs a small sampling time to obtain good results. The proposed scheme to improve the modulation scheme is based on a Discrete Space Vector with virtual vectors to obtain a better approximation of the optimal vectors for use in the algorithm. The proposal improves the conventional scheme at a high sampling time (200 μs), obtaining a THD less than 2% in the load current, decreases the noise created by the conventional scheme, and provides a fixed switching frequency. Experimental tests demonstrate the correct operation of the proposed scheme.

Electrofuels in a circular economy: A systems approach towards net zero

Decarbonising the hard-to-abate sectors will be necessary in realising a future net-zero economy. Electrofuels store electricity as low carbon energy vectors such as hydrogen or methane which can be used in areas where electrification is not ideal, and as such can facilitate decarbonisation of sectors such as transport, agriculture, and wastewater treatment. In this study, the production of electrofuels was analysed at an industrial site by storing renewable electricity as green hydrogen produced using electrolysis. The analysis highlighted the need for scale in hydrogen production. The cost of hydrogen was calculated at €8.92/kg when a 122 kW electrolyser operated solely on curtailed electricity generated from the industry site was situated at a 65,000 person equivalent municipal wastewater treatment plant. A subsequent integrated and circular approach to electrofuels production was investigated. The oxygen by-product from electrolysis could be utilised for wastewater aeration and reduce the annual electricity usage at the wastewater treatment plant by 3.6%. Furthermore, the carbon dioxide in biogas generated from sewage sludge could be converted to methane through a Sabatier reaction (4H2 + CO2 → CH4 + 2H2O) as a means of carbon capture and utilisation. The hydrogen produced from the 122 kW electrolyser could convert only 40% of the total carbon dioxide (within the biogas) in a biomethanation process, again supporting the argument for larger electrolyser systems with increased hydrogen production. Pyrolysis of digestate to produce biochar was investigated as a negative emissions technology. If pyrolysis is coupled with anaerobic digestion of feedstocks within 10 km of the industry site savings of 42.7 kt CO2/a could be achieved. In essence, a circular economy approach to electrofuel production could integrate existing electrical, gas and water infrastructure, whilst treating waste, improving the environment, decarbonising agriculture, and storing energy in the form of new low carbon energy vectors for use in heavy transport. Such an approach is vital to progressing future net-zero strategies, however future emissions accountancy processes must adapt to facilitate the benefits of a circular economy.

Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery

Viral vector gene therapy is an attractive strategy to treat hearing loss. Since hearing loss is due to a variety of pathogenic signaling cascades in distinct cells, viral vectors that can express large or multiple genes in a cell-type specific manner are needed. Helper-dependent adenoviral vectors (HdAd) are safe viral vectors with a large packaging capacity (-36 kb). Despite the potential of HdAd, its use in the inner ear is largely unexplored. Therefore, to evaluate the utility of HdAd for inner ear gene therapy, we created two HdAd vectors that use distinct cellular receptors for transduction: HdAd Serotype Type 5 (HdAd5), the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35, the human CD46+ receptor (hCD46). We delivered these vectors through the round window (RW) or scala media in CBA/J, C57Bl6/J and hCD46 transgenic mice. Immunostaining in conjunction with confocal microscopy of cochlear sections revealed that multiple cell types were transduced using HdAd5 and HdAd 5/35 in all mouse models. Delivery of HdAd5 via RW in the C57Bl/6 J or CBA/J cochlea resulted in transduced mesenchymal cells of the peri‑lymphatic lining and modiolar region while scala media delivery resulted in transduction of supporting cells and inner hair cells. Hd5/35 transduction was CD46 dependent and RW delivery of HdAd5/35 in the hCD46 mouse model resulted in a similar transduction pattern as HdAd5 in the peri‑lymphatic lining and modiolar region in the cochlea. Our data indicate that HdAd vectors are promising vectors for use in inner ear gene therapy to treat some causes of hearing loss.

Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots.

Many plant viruses have been engineered into vectors for use in functional genomics studies, expression of heterologous proteins, and, most recently, gene editing applications. The use of viral vectors overcomes bottlenecks associated with mutagenesis and transgenesis approaches often implemented for analysis of gene function. There are several engineered viruses that are demonstrated or suggested to be useful in maize through proof-of-concept studies. However, foxtail mosaic virus (FoMV), which has a relatively broad host range, is emerging as a particularly useful virus for gene function studies in maize and other monocot crop or weed species. A few clones of FoMV have been independently engineered, and they have different features and capabilities for virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX) of proteins. In addition, FoMV can be used to deliver functional guide RNAs in maize and other plants expressing the Cas9 protein, demonstrating its potential utility in virus-induced gene editing applications. There is a growing number of studies in which FoMV vectors are being applied for VIGS or VOX in maize and the vast majority of these are related to maize-microbe interactions. In this review, we highlight the biology and engineering of FoMV as well as its applications in maize-microbe interactions and more broadly in the context of the monocot functional genomics toolbox.

New temperature-switchable acyl homoserine lactone-regulated expression vector.

Bacterial expression systems play an indispensable role in the biosynthesis of recombinant proteins. Different proteins and the tasks associated with them may require different systems. The purpose of this work is to make an expression vector that allows switching on and off the expression of the target gene during cell incubation. Several expression vectors for use in Escherichiacoli cells were developed using elements of the luxR/luxI type quorum sensing system of psychrophilic bacterium Aliivibrio logei. These vectors contain A.logei luxR2 and (optionally) luxI genes and LuxR2-regulated promoter, under the control of which a target gene is intended to be inserted. The synthesis of the target protein depends directly on the temperature: gene expression starts when the temperature drops to 22°C and stops when it rises to 37°C, which makes it possible to fix the desired amount of the target protein in the cell. At the same time, the expression of the target gene at a low temperature depends on the concentration of the autoinducer (L-homoserine N-(3-oxohexanoyl)-lactone, AI) in the culture medium in a wide range from 1nM to 10μM, which makes it possible to smoothly regulate the rate of target protein synthesis. Presence of luxI in the vector provides the possibility of autoinduction. Constructed expression vectors were tested with gfp, ardA, and ardB genes. At maximum, we obtained the target protein in an amount of up to 33% of the total cellular protein. KEY POINTS: • A.logei quorum sensing system elements were applied in new expression vectors • Expression of target gene is inducible at 22°C and it is switched off at 37°C • Target gene expression at 22°C is tunable by use different AI concentrations.

Boosters for adeno-associated virus (AAV) vector (r)evolution

Adeno-associated virus (AAV) is one of the most exciting and most versatile templates for engineering of gene-delivery vectors for use in human gene therapy, owing to the existence of numerous naturally occurring capsid variants and their amenability to directed molecular evolution. As a result, the field has witnessed an explosion of novel “designer” AAV capsids and ensuing vectors over the last two decades, which have been isolated from comprehensive capsid libraries generated through technologies such as DNA shuffling or peptide display, and stratified under stringent positive and/or negative selection pressures. Here, we briefly highlight a panel of recent, innovative and transformative methodologies that we consider to have exceptional potential to advance directed AAV capsid evolution and to thereby accelerate AAV vector revolution. These avenues comprise original technologies for (i) barcoding and high-throughput screening of individual AAV variants or entire capsid libraries, (ii) selection of transduction-competent AAV vectors on the DNA level, (iii) enrichment of expression-competent AAV variants on the RNA level, as well as (iv) high-resolution stratification of focused AAV capsid libraries on the single-cell level. Together with other emerging AAV engineering stratagems, such as rational design or machine learning, these pioneering techniques promise to provide an urgently needed booster for AAV (r)evolution.

Biotechnological Innovations and Therapeutic Application of Pediococcus and Lactic Acid Bacteria: The Next-Generation Microorganism.

Lactic acid bacteria represent a worthwhile organism within the microbial consortium for the food sector, health, and biotechnological applications. They tend to offer high stability to environmental conditions, with an indicated increase in product yield, alongside their moderate antimicrobial activity. Lack of endotoxins and inclusion bodies, extracellular secretion, and surface display with other unique properties, are all winning attributes of these Gram-positive lactic acid bacteria, of which, Pediococcus is progressively becoming an attractive and promising host, as the next-generation probiotic comparable with other well-known model systems. Here, we presented the biotechnological developments in Pediococcal bacteriocin expression system, contemporary variegated models of Pediococcus and lactic acid bacteria strains as microbial cell factory, most recent applications as possible live delivery vector for use as therapeutics, as well as upsurging challenges and future perspective. With the radical introduction of artificial intelligence and neural network in Synthetic Biology, the microbial usage of lactic acid bacteria as an alternative eco-friendly strain, with safe use properties compared with the already known conventional strains is expected to see an increase in various food and biotechnological applications in years to come as it offers better hope of safety, accuracy, and higher efficiency.

A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells.

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.

VikAD, a Vika site-specific recombinase-based system for efficient and scalable helper-dependent adenovirus production

Recombinant viral vectors have become integral tools for basic in vivo research applications. Helper-dependent adenoviral (HdAd) vectors have a large packaging capacity of ∼36 kb of DNA that mediate long-term transgene expression in vitro and in vivo. The large carrying capacity of HdAd enables basic research or clinical applications requiring the delivery of large genes or multiple transgenes, which cannot be packaged into other widely used viral vectors. Currently, common HdAd production systems use an Ad helper virus (HV) with a packaging signal (Ψ) that is flanked by either loxP or FRT sites, which is excised in producer cells expressing Cre or Flp recombinases to prevent HV packaging. However, these production systems prevent the use of HdAd vectors for genetic strategies that rely on Cre or Flp recombination for cell-type-specific expression. To overcome these limitations, we developed the VikAD production system, which is based on producer cells expressing the Vika recombinase and an HV that contains a Ψ flanked by vox sites. The availability of this production system will greatly expand the utility and flexibility of HdAd vectors for use in research applications to monitor and manipulate cellular activity with increased specificity.

AFM study of organic ligand packing on gold for nanoparticle drug delivery applications

Gold nanoparticles (AuNPs) functionalized with polyethylene glycol (PEG) and peptide ligands are promising drug delivery vectors for use in cancer therapy. However, to control the interaction of the ligands with blood proteins and specific cell receptors, more needs to be known about the arrangement and properties of these grafted ligands. This issue is examined here principally with atomic force microscopy (AFM) for ligands grafted to both gold films and gold nanoparticles. Macro-level sessile drop measurements on the films indicate that, in dilute solutions, the PEG and peptide ligands exhibit similar affinity to the gold surface. However, at the nano-level, AFM experiments show differences in the morphology and cohesion of the adsorbed ligand films. Differences in tip/surface adhesion are also measured, indicative of variations of hydrophilicity for the top surface of these ligand films. Analysis of force-separation curves suggests a low packing density of PEG ligands. AFM investigations of functionalized AuNPs shows that the ligands improve dispersion and modify the tip/surface adhesion behaviour on the nanoparticles. However, AFM images of as-synthesised citric-capped and functionalized AuNPs gave similar nanoparticle diameters. Analysis of the AFM tip tapping on the AuNPs suggests that Tapping-AFM is not suitable for detection of low packing density ligands.

Automated evaluation of Cr-III coated parts using Mask RCNN and ML methods

In this study, chrome coatings were carried out using a Cr-III electroplating bath. The coated parts were classified depending on their appearance. A new approach was developed to classify the coated parts automatically using artificial intelligence methods. Mask RCNN and machine learning (ML) methods such as Multilayer Perceptron (MLP), Support Vector Classifier (SVC), Gaussian Process (GP), K-nearest Neighbors (KNN), XGBoost, and Random Forest Classifier (RFC) were used together. Mask RCNN was used to clean the coated parts from the redundant data. The extracted data were flattened and converted to the row vectors for use as input in ML methods. ML algorithms were used to classify the coated parts as “Pass” and “Fail”. The classification accuracy was checked with the leave one out (loo) cross-validation method. RFC method gave the highest accuracy, 0.83, and F1 score, 0.88. The accuracy of Mask RCNN was checked using a dataset of separated validation images. It was observed that extracting the unnecessary data from the images increased the accuracy exceedingly. Moreover, the method exhibits a high potential to keep the parameters of the electroplating process under control.

An efficient papaya leaf distortion mosaic potyvirus vector for virus-induced gene silencing in papaya

Papaya (Carica papaya L.) is regarded as an excellent model for genomic studies of tropical trees because of its short generation time and its small genome that has been sequenced. However, functional genomic studies in papaya depend on laborious genetic transformations because no rapid tools exist for this species. Here, we developed a highly efficient virus-induced gene silencing (VIGS) vector for use in papaya by modifying an artificially attenuated infectious clone of papaya leaf distortion mosaic virus (PLDMV; genus: Potyvirus), PLDMV-E, into a stable Nimble Cloning (NC)-based PLDMV vector, pPLDMV-NC, in Escherichia coli. The target fragments for gene silencing can easily be cloned into pPLDMV-NC without multiple digestion and ligation steps. Using this PLDMV VIGS system, we silenced and characterized five endogenous genes in papaya, including two common VIGS marker genes, namely, phytoene desaturase, Mg-chelatase H subunit, putative GIBBERELLIN (GA)-INSENSITIVE DWARF1A and 1B encoding GA receptors; and the cytochrome P450 gene CYP83B1, which encodes a key enzyme involved in benzylglucosinolate biosynthesis. The results demonstrate that our newly developed PLDMV VIGS vector is a rapid and convenient tool for functional genomic studies in papaya.

PET expression vector customized for efficient seamless cloning.

Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis.

A Hybrid Biological-Biomaterial Vector for Antigen Delivery.

A hybrid biological-biomaterial vector composed of a biocompatible polymeric biomaterial coating and an Escherichia coli core was designed and developed for antigen delivery. It provides a unique and efficient mechanism to transport antigens (protein or genetic) via different mechanisms of vector design that include antigen cellular localization (cytoplasm, periplasm, cellular surface) and nonnative functionalities that assist in antigen delivery. Based on a variety of E. coli strain development and polymer chemistry tools, the hybrid vector can be constructed into a number of formats for the purpose of optimized uptake and processing by antigen presenting cells, serving as the basis for a potent subsequent immune response. This chapter serves to outline a protocol for assembling a hybrid biological-biomaterial vector for use as a vaccine delivery system.

Alteration of gammaretroviral vector integration patterns by insertion of histone and leucine zipper into integrase.

Retroviral vectors show long-term gene expression in gene therapy through the integration of transgenes into the human cell genome. Murine leukemia virus (MLV), a well-studied gammaretrovirus, has been often used as a representative retroviral vector. However, frequent integrations of MLV-based vectors into transcriptional start sites (TSSs) could lead to the activation of oncogenes by enhancer effects of the genetic components within the vectors. Therefore, the MLV integration preference for TSSs limits its wider use in clinical applications. To reduce the integration preference of MLV-based vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted histones and leucine zippers, having DNA-binding property, into internal sites of MLV integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild-type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer MLV-based retroviral vectors for use in clinical applications.

Main Features of DNA-Based Vectors for Use in Lactic Acid Bacteria and Update Protocols.

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.

Neutralisation of adeno-associated virus transduction by human vitreous humour.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system

BackgroundRecombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as “GoldenBac”.ResultsThis method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2–15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein.ConclusionsThis robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.