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Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases. Golden Gate assembly of 10 diterpene synthases (diTPS) and 4 cytochrome CYP oxygenases (CYPs) from diverse plant species in the yeast Saccharomyces cerevisiae leads to the production of more than 200 distinct diterpenoids. 132 of these diterpenoids are new-to-Nature indicating that combinatorial assembly of diTPS and CYPs in yeast is a promising approach to generate diverse libraries of diterpenoids in high-throughput. [Display omitted] • The biosynthesis of diterpenoids is modular. • Modularity allows the combinatorial assembly of diterpene synthases and cytochrome P450 oxygenases. • 10 diterpene synthases and 4 CYPs from different plants were combined using Golden Gate cloning and expressed in yeast. • In total more than 200 diterpenoids could be detected. • 162 of these diterpenoids are new-to-Nature. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa) is a staple food consumed by the majority of the world’s population. Climate change, however, has created a significant threat to our food security as it posed severe effects on rice production. The emergence of genome editing technology has opened a new era in crop improvement. Hence, this study aims to develop the CRISPR/Cas9 construct of drought tolerance for O. sativa subsp. indica cv. IR64 using Golden Gate cloning method. For this purpose, the generation of CRISPR/Cas9 constructs involved several stages, i.e., characterization of SUMO E2-Conjugating Enzyme (OsSCE1) gene, single-guide RNA (sgRNA) design and vector construction. FGENESH, GeneMarkS, InterProScan, and Blast2GO programmes – were used for the OsSCE1 gene characterisation. The putative OsSCE1 gene isolated from IR64 was then verified by sequencing, and the gene was 585 bp long and showed 99% identity with O. sativa on chromosome 10. In silico analysis concluded the gene is involved in abiotic stress regulation. The 20 bp sgRNA was designed manually with the aid of gRNA prediction programmes including CCTop, and Benchling. The virtual vector was validated using the Golden Gate Cloning approach and later confirmed through sequencing. The assembly involved separate vectors containing the OsSCE1 sgRNA sequence, plant selectable marker, and Cas9 cassette to construct standardised elements for hierarchical modular cloning (MoClo). This study demonstrated that our format, as the gene insertion are achievable, resulting in a speedier and more efficient assembly process which may contribute to improve drought tolerance in indica rice. Further study on the Agrobacterium-mediated transformation using the developed construct may be conducted to determine the efficacy of knocking out candidate genes in improving drought tolerance ability O. sativa

Rice (Oryza sativa) is a staple food consumed by the majority of the world's population. Climate change, however, has created a significant threat to our food security as it posed severe effects on rice production. The emergence of genome editing technology has opened a new era in crop improvement. Hence, this study aims to develop the CRISPR/Cas9 construct of drought tolerance for O. sativa subsp. indica cv. IR64 using Golden Gate cloning method. For this purpose, the generation of CRISPR/Cas9 constructs involved several stages, i.e., characterization of SUMO E2-Conjugating Enzyme (OsSCE1) gene, single-guide RNA (sgRNA) design and vector construction. FGENESH, GeneMarkS, InterProScan, and Blast2GO programmes -- were used for the OsSCE1 gene characterisation. The putative OsSCE1 gene isolated from IR64 was then verified by sequencing, and the gene was 585 bp long and showed 99% identity with O. sativa on chromosome 10. In silico analysis concluded the gene is involved in abiotic stress regulation. The 20 bp sgRNA was designed manually with the aid of gRNA prediction programmes including CCTop, and Benchling. The virtual vector was validated using the Golden Gate Cloning approach and later confirmed through sequencing. The assembly involved separate vectors containing the OsSCE1 sgRNA sequence, plant selectable marker, and Cas9 cassette to construct standardised elements for hierarchical modular cloning (MoClo). This study demonstrated that our format, as the gene insertion are achievable, resulting in a speedier and more efficient assembly process which may contribute to improve drought tolerance in indica rice. Further study on the Agrobacterium-mediated transformation using the developed construct may be conducted to determine the efficacy of knocking out candidate genes in improving drought tolerance ability O. sativa. [ABSTRACT FROM AUTHOR]

Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, Chlamydomonas reinhardtii has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs to be optimized. In many model organisms, viral promoters are used to drive transgene expression at high levels. However, no viruses are known to infect Chlamydomonas, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of Chlamydomonas reinhardtii field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in Chlamydomonas. We used ble, NanoLUC, and mCherry as reporter genes, and three native benchmark promoters as controls. None of the viral promoters drove expression of any reporter gene beyond background. During our study, we found that mCherry variants are produced by alternative in-frame translational start sites in Chlamydomonas. We show that this problem can be overcome by mutating the responsible methionine codons to codons for leucine and by using the 5′-UTR of βTUB2 instead of the 5′-UTRs of PSAD or RBCS2. Apparently, the βTUB2 5′-UTR promotes the use of the first start codon. This could be mediated by the formation of a stem-loop between sequences of the βTUB2 5′-UTR and sequences downstream of the first AUG in the mCherry reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning. [ABSTRACT FROM AUTHOR]

Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression. [ABSTRACT FROM AUTHOR]

With the advent of multiple omics and Genome-Wide Association Studies (GWAS) technology, genome-scale functional analysis of candidate genes is to be conducted in diverse plant species. Construction of plant binary expression vectors is the prerequisite for gene function analysis. Therefore, it is of significance to develop a set of plant binary expression vectors with highly efficient, inexpensive, and convenient cloning method, and easy-to-use in screening of positive recombinant in Escherichia coli. In this study, we developed a set of plant binary expression vectors, termed pBTR vectors, based on Golden Gate cloning using BsaI restriction site. Foreign DNA fragment of interest (FDI) can be cloned into the destination pBTR by onestep digestion-ligation reaction in a single tube, and even the FDI contains internal BsaI site(s). Markedly, in one digestion-ligation reaction, multiple FDIs (exemplified by cloning four soybean Glyma.02g025400, Glyma.05g201700, Glyma.06g165700, and Glyma.17g095000 genes) can be cloned into the pBTR vector to generate multiple corresponding expression constructs (each expression vector carrying an FDI). In addition, the pBTR vectors carry the visual marker, a brightness monomeric red fluorescent protein mScarlet-I, that can be observed with the unaided eye in screening of positive recombinants without the use of additional reagents/equipment. The reliability of the pBTR vectors was validated in plants by overexpression of AtMyb75/PAP1 in tomato and GUSPlus in soybean roots via Agrobacterium rhizogenes-mediated transformation, promoter activity analysis of AtGCSpro in Arabidopsis via A. tumefaciens-mediated transformation, and protein subcellular localization of the Vitis vinifera VvCEB1opt in tobacco, respectively. These results demonstrated that the pBTR vectors can be used in analysis of gene (over) expression, promoter activity, and protein subcellular localization. These vectors will contribute to speeding up gene function analysis and the process of plant molecular breeding. [ABSTRACT FROM AUTHOR]

Efficient expression of multiple genes is critical to yeast metabolic engineering for the bioproduction of bulk and fine chemicals. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. 2A viral peptides enable the cotranslation of multiple proteins from a single mRNA by ribosomal skipping. However, the wide adaptation of 2A viral peptides for polycistronic‐like gene expression in yeast awaits in‐depth characterizations. Additionally, a one‐step assembly of such a polycistronic‐like system is highly desirable. To this end, we have developed a modular cloning (MoClo) compatible 2A peptide‐based polycistronic‐like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi‐, tri‐, and quad‐cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides: E2A from equine rhinitis B virus, P2A from porcine teschovirus‐1, and O2A from Operophtera brumata cypovirus‐18. Applying the polycistronic‐like system to produce geraniol, a valuable industrial compound, resulted in comparable or higher titers than using conventional monocistronic constructs. In summary, this highly‐characterized polycistronic‐like gene expression system is another tool to facilitate multigene expression for metabolic engineering in yeast. [ABSTRACT FROM AUTHOR]

In vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-expression of defined organelle markers (OM). Several marker sets are available but, so far, the procedure requires successful co-transformation of POI and OM into the same cell and/or several cloning steps. We developed a set of vectors containing markers for basic cell organelles that enables the insertion of the gene of interest (GOI) by a single cloning step using the Golden Gate cloning approach and resulting in POI–GFP fusions. The set includes markers for plasma membrane, tonoplast, nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, plastids, and mitochondria, all labelled with mCherry. Most of them were derived from well-established marker sets, but those localized in plasma membrane and tonoplast were improved by using different proteins. The final vectors are usable for localization studies in isolated protoplasts and for transient transformation of leaves of Nicotiana benthamiana. Their functionality is demonstrated using two enzymes involved in biosynthesis of jasmonic acid and located in either plastids or peroxisomes. [ABSTRACT FROM AUTHOR]

Combinatorial library-based metabolic engineering approaches allow lower cost and faster strain development. We developed a genetic toolbox EXPRESS YALI for combinatorial engineering of the oleaginous yeast Yarrowia lipolytica. The toolbox enables consecutive rounds of engineering, where up to three combinatorially assembled gene expression cassettes can be integrated into each yeast clone per round. The cassettes are integrated into distinct intergenic sites or an open reading frame of a target gene if a simultaneous gene knockout is desired. We demonstrate the application of the toolbox by optimizing the Y. lipolytica to produce the red beet color betanin via six consecutive rounds of genome editing and screening. The library size varied between 24 and 360. Library screening was facilitated by automated color-based colony picking. In the first round, betanin pathway genes were integrated, resulting in betanin titer of around 20 mg/L. Through the following five consecutive rounds, additional biosynthetic genes were integrated, and the precursor supply was optimized, resulting in a titer of 70 mg/L. Three beta-glucosidases were deleted to prevent betanin deglycosylation, which led to a betanin titer of 130 mg/L in a small scale and a titer of 1.4 g/L in fed-batch bioreactors. The EXPRESS YALI toolbox can facilitate metabolic engineering efforts in Y. lipolytica (available via AddGene Cat. Nr. 212682-212704, Addgene kit ID # 1000000245)., Competing Interests: Declaration of competing interest IB declares a competing interest as a co-inventor of a patent application related to the study. Other authors declare that they have no known competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Synthetic Biology is revolutionizing biological research by introducing principles of mechanical engineering, including the standardization of genetic parts and standardized part assembly routes. Both are realized in the Modular Cloning (MoClo) strategy. MoClo allows for the rapid and robust assembly of individual genes and multigene clusters, enabling iterative cycles of gene design, construction, testing, and learning in short time. This is particularly true if generation times of target organisms are short, as is the case for the unicellular green alga Chlamydomonas reinhardtii. Testing a gene of interest in Chlamydomonas with MoClo requires two assembly steps, one for the gene of interest itself and another to combine it with a selection marker. To reduce this to a single assembly step, we constructed five new destination vectors. They contain genes conferring resistance to commonly used antibiotics in Chlamydomonas and a site for the direct assembly of basic genetic parts. The vectors employ red/white color selection and, therefore, do not require costly compounds like X-gal and IPTG. mCherry expression is used to demonstrate the functionality of these vectors. [ABSTRACT FROM AUTHOR]

Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. While currently offering the most affordable source of synthetic DNA, oligo pools also come with limitations such as a maximum synthesis length (approximately 350 bases), a higher error rate compared to alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single-guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Golden Gate cloning has become one of the most popular DNA assembly techniques. Its modular and hierarchical structure allows the construction of complex DNA fragments. Over time, Golden Gate cloning allows for the creation of a repository of reusable parts, reducing the cost of frequent sequence validation. However, as the number of reactions and fragments increases, so does the cost of consumables and the potential for human error. Typically, Golden Gate reactions are performed in volumes of 10-25 μL. Recent technological advances have led to the development of liquid handling robots that use sound to transfer liquids in the nL range from a source plate to a target plate. These acoustic dispensers have become particularly popular in the field of synthetic biology. The use of this technology allows miniaturization and parallelization of molecular reactions in a tip-free manner, making it sustainable by reducing plastic waste and reagent usage. Here, we provide a step-by-step protocol for performing and parallelizing Golden Gate cloning reactions in 1 μL total volume., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences and results in synthesis failure or delays by DNA synthesis vendors. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible overhangs. Then synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate assembly., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Golden Gate cloning technique is used to assemble DNA parts into higher-order assemblies. Individual parts containing compatible overhangs generated by type IIS restriction enzymes are joined together using DNA ligase. The technique enables users to assemble custom transcription units (TUs) for a wide array of experimental assays. Several Golden Gate cloning systems have been developed; however, they are typically used with a narrow range of organisms. Here we describe the Multi-Kingdom (MK) cloning system that allows users to generate DNA plasmids for use in a broad range of organisms., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Researchers have dedicated efforts to refining genetic part assembly techniques, responding to the demand for complex DNA constructs. The optimization efforts, targeting enhanced efficiency, fidelity, and modularity, have yielded streamlined protocols. Among these, Golden Gate cloning has gained prominence, offering a modular and hierarchical approach for constructing complex DNA fragments. This method is instrumental in establishing a repository of reusable parts, effectively reducing the costs and proving highly valuable for high-throughput DNA assembly projects. In this review, we delve into the main protocol of Golden Gate cloning, providing refined insights to enhance protocols and address potential challenges. Additionally, we perform a thorough evaluation of the primary modular cloning toolkits adopted by the scientific community. The discussion includes an exploration of recent advances and challenges in the field, providing a comprehensive overview of the current state of Golden Gate cloning., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox. Functional DNA templates composed of multiple parts such as promoter, ribosomal binding site (RBS), coding sequence, and terminator are produced in vitro in a one-pot Golden Gate assembly reaction followed by polymerase chain reaction (PCR) amplification. We demonstrate assembly, cell-free testing of promoter and RBS combinations, as well as characterization of a repressor-promoter pair. By eliminating time-consuming transformation and cloning steps in cells and by taking advantage of modular cloning toolboxes, our cell-free prototyping workflow can produce data for large numbers of new assembled constructs within a single day., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Prokaryotes use CRISPR-Cas systems to interfere with viruses and other mobile genetic elements. CRISPR arrays comprise repeated DNA elements and spacer sequences that can be engineered for custom target sites. These arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) that undergo maturation steps to form individual CRISPR RNAs (crRNAs). Each crRNA contains a single spacer that identifies the target cleavage site for a large variety of Cas protein effectors. Precise manipulation of spacer sequences within CRISPR arrays is crucial for advancing the functionality of CRISPR-based technologies. Here, we describe a protocol for the design and creation of a minimal, plasmid-based CRISPR array to enable the expression of specific, synthetic crRNAs. Plasmids contain entry spacer sequences with two type IIS restriction sites and Golden Gate cloning enables the efficient exchange of these spacer sequences. Factors that influence the compatibility of the CRISPR arrays with native or recombinant Cas proteins are discussed., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents.

Non-homologous end-joining (NHEJ)-mediated random integration in Yarrowia lipolytica has been demonstrated to be an effective strategy for screening hyperproducer strains. However, there was no multigene assembly method applied for NHEJ integration, which made it challenging to construct and integrate metabolic pathways. In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in Y. lipolytica. By optimizing key factors, including the amounts of ligase and the reaction cycles, the assembly efficiency of 4, 7, and 10 fragments reached up to 90, 75, and 50%, respectively. This YALIcloneNHEJ system was subsequently applied for the overproduction of the sesquiterpene (-)-α-bisabolol by constructing a biosynthesis route and enhancing the flux in the mevalonate pathway. The resulting strain produced 4.4 g/L (-)-α-bisabolol, the highest titer reported in yeast to date. Our study expands the toolbox of metabolic engineering and is expected to enable a highly efficient production of various terpenoids.

Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained transgenic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding. [ABSTRACT FROM AUTHOR]

Abstract Background Recombinant 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”. Results This 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. Conclusions This 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.

CRISPR-Cas9 technology has become an essential tool for plant genome editing. Recent advancements have significantly improved the ability to target multiple genes simultaneously within the same genetic background through various strategies. Additionally, there has been significant progress in developing methods for inducible or tissue-specific editing. These advancements offer numerous possibilities for tailored genome modifications. Building upon existing research, we have developed an optimized and modular strategy allowing the targeting of several genes simultaneously in combination with the synchronized expression of the Cas9 endonuclease in the egg cell. This system allows significant editing efficiency while avoiding mosaicism. In addition, the versatile system we propose allows adaptation to inducible and/or tissue-specific edition according to the promoter chosen to drive the expression of the Cas9 gene. Here, we describe a step-by-step protocol for generating the binary vector necessary for establishing Arabidopsis edited lines using a versatile cloning strategy that combines Gateway ® and Golden Gate technologies. We describe a versatile system that allows the cloning of as many guides as needed to target DNA, which can be multiplexed into a polycistronic gene and combined in the same construct with sequences for the expression of the Cas9 endonuclease. The expression of Cas9 is controlled by selecting from among a collection of promoters, including constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that, sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination reaction is required to assemble the promoter, the zCas9 coding sequence, and the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and finally assembled according to the Multisite Gateway ® Technology. Here, we detail the process to express zCas9 under the control of egg cell promoter fused to enhancer sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains and transmembrane region protein genes ( MCTP3 and MCTP4 , respectively at3g57880 and at1g51570), using one or two sgRNA per gene. Key features • A simple method for Arabidopsis edited lines establishment using CRISPR-Cas9 technology • Versatile cloning strategy combining various technologies for convenient cloning (Gateway ® , Golden Gate) • Multigene targeting with high efficiency., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

Aims: Expression of recombinant proteins across a range of different host organisms has profound contribution to the advancement in biotechnology. In this study, we aimed to construct a highly versatile broad host range (BHR) expression vector, designated as pYL101C. Methodology and results: The Golden Gate cloning approach was used to construct pYL101C. Key features of pYL101C include a strong integron promoter (PINTc), a BHR pBBR1 origin of replication (ori), gentamycin resistance gene (GmR) as a selectable marker and a multiple cloning site (MCS) downstream of the promoter for easy-cloning purpose. To verify the functionality of pYL101C, we cloned the superfolder green fluorescent protein (sfGFP) reporter gene into pYL101C and transferred the resultant recombinant plasmid pYL101C::sfGFP into various Gram-negative bacteria. Transformants obtained stably expressed strong green fluorescence under blue light excitation even without selection after four passages. Conclusion, significance and impact of study: The constructed BHR expression vector, pYL101C and recombinant pYL101C::sfGFP are stable and can be used to monitor the presence of Gram-negative bacteria, such as endophytes and pathogens in their hosts and environment. [ABSTRACT FROM AUTHOR]

In vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-expression of defined organelle markers (OM). Several marker sets are available but, so far, the procedure requires successful co-transformation of POI and OM into the same cell and/or several cloning steps. We developed a set of vectors containing markers for basic cell organelles that enables the insertion of the gene of interest (GOI) by a single cloning step using the Golden Gate cloning approach and resulting in POI–GFP fusions. The set includes markers for plasma membrane, tonoplast, nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, plastids, and mitochondria, all labelled with mCherry. Most of them were derived from well-established marker sets, but those localized in plasma membrane and tonoplast were improved by using different proteins. The final vectors are usable for localization studies in isolated protoplasts and for transient transformation of leaves of Nicotiana benthamiana. Their functionality is demonstrated using two enzymes involved in biosynthesis of jasmonic acid and located in either plastids or peroxisomes.

The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi. [ABSTRACT FROM AUTHOR]

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.

Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained transgenic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding.

Abstract Background Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Results Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. Conclusion We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs. [ABSTRACT FROM AUTHOR]

As a plant genetic engineer, Agrobacterium tumefaciens utilizes phenolic compounds, such as acetosyringone, to activate its virulence genes during the infection and transformation process. In this study, two novel broad-host-range (BHR) acetosyringone-inducible expression vectors were constructed. Unlike Agrobacterium binary vectors which are developed for plant transformation, these vectors were designed for heterologous gene expression in A. tumefaciens. To this end, an acetosyringone-inducible virB promoter (PvirB) was placed upstream of a Gateway® cassette for plasmid vector pASE1 and Type IIS recognition sites, BpiI for plasmid vector pASE2, thus allowing the PvirB to drive the expression of the insert, which may be cloned into pASE1 and pASE2 via Gateway® LR Cloning and Golden Gate Cloning approaches, respectively. Here, we tested the functionality and inducibility of pASE1 and pASE2 vectors by expressing the reporter gene superfolder green fluorescent protein (sfGFP) in the presence of acetosyringone. The expression of the reporter gene sfGFP after induction by acetosyringone was verified by Western blotting. Interestingly, the presence of the attB1 site in pASE1 imposed a tight regulation on the inducibility of the PvirB. This is the first report that indicates that an attB1 site may hinder downstream gene expression in a bacterium. We envision that pASE2 can be used to express key virulence genes to enhance plant transformation and advance the genetic studies of Agrobacterium. [ABSTRACT FROM AUTHOR]

Background: Recombinant 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". Results: This 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. Conclusions: This 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. [ABSTRACT FROM AUTHOR]

In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. We show that single chain variable fragment (scFv) libraries with adequate qualities can readily be cloned in a 'scar-less' manner and that the isolation of antigen-specific antibodies from immunized chickens is feasible within three selection rounds. Moreover, we demonstrate the general applicability of this method by rapidly constructing and panning VHH single domain antibody phage display libraries from immunized Llama repertoires. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.

Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial. This protocol outlines how to use computational predictions and synthetic biology approaches to design, construct, and validate synthetic sRNA functionality for their application in bacteria. The computational tool, SEEDling, matches the optimal seed region with the user-selected sRNA scaffold for repression of target mRNAs. The synthetic sRNAs are assembled using Golden Gate cloning and their functionality is subsequently validated. The protocol uses the acrA mRNA as an exemplary proof-of-concept target in Escherichia coli. Since AcrA is part of a multidrug efflux pump, acrA repression can be revealed by assessing oxacillin susceptibility in a phenotypic screen. However, in case target repression does not result in a screenable phenotype, an alternative validation of synthetic sRNA functionality based on a fluorescence reporter is described., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo. Here, we improved the sensitivity of this technology and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high-throughput screening of candidate proteins we developed a Golden Gate-based vector toolkit (PlaMinGo). As a result of these improvements, dual targeting could be detected for a number of proteins that had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach based on the immutans mutant. [ABSTRACT FROM AUTHOR]

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo. Here, we improved this technology with respect to sensitivity and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high throughput screening of candidate proteins we developed a Golden Gate-based vector toolkit (PlaMinGo). As a result of these improvements, dual targeting could be detected for a number of proteins, which had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach based on the immutans mutant. [ABSTRACT FROM AUTHOR]

In the present study, a donor plasmid derived from pUC19 and two recipient plasmids, which had been modified from the donor plasmid and contained the red fluorescence protein gene mCherry as a reporter gene downstream of the hybrid tac promoter with the −35 region deletion mutation, were constructed. The complete genome sequence of coxsackievirus A10 downstream of the T7 promoter was divided into 7 fragments and synthesized by overlap extension PCR and the DNAworks program. Using the Golden Gate cloning strategy, the 7 fragments were then cloned into the donor plasmid and transferred to the recipient plasmid upstream of the deletion mutation tac promoter in a defined order and orientation without any deletions or insertions at the junction sites. Because the −35 region of the tac promoter was introduced into the 3′ end of the last fragment during construction, the hybrid promoter was reconstructed to promote expression of mCherry , which facilitated the selection of colonies with the complete genome of coxsackievirus A10 to generate an infectious cDNA clone via reverse genetic engineering. • The complete cDNA genome of CVA10 was de novo synthesized based on overlap extension PCR and the DNAworks program. • The complete cDNA genome of CVA10 was assembled based on Golden Gate cloning. • The CVA10 strain was successfully rescued from Vero cells. • Blue-white screening and red-white screening were performed in the synthesis and assembly steps, respectively. • A small DNA fragment was assembled in pAC; a large DNA fragment was assembled in pRC. [ABSTRACT FROM AUTHOR]

Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization. [ABSTRACT FROM AUTHOR]

Here we present an improved strategy for producing T-cell receptor (TCR)-expressing retroviral vectors using a Golden Gate cloning strategy. This method takes advantage of the modular nature of TCR genes by directly amplifying TCR α and β variable regions from RNA or cDNA, then cloning and fusing them with their respective constant region genes resident in a retroviral TCR expression vector. Our one-step approach greatly streamlines the TCR vector production process in comparison to the traditional three-step procedure that typically involves cloning whole TCR genes, producing a TCR expression cassette, and constructing a retroviral construct. To date, we have generated TCR vectors that transferred seven functional human/rhesus macaque TCRs into primary T cells. The approach also holds promise for the assembly of other genes with defined variable regions, such as immunoglobulins.

Summary: Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post‐translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process. Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning. This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression. We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time‐ and cost‐effective manner. [ABSTRACT FROM AUTHOR]

Background: Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Results: Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. Conclusion: We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires. [ABSTRACT FROM AUTHOR]

Background: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P. pastoris. Methods: With the GoldenPiCS system, we provide a flexible modular system for advanced strain engineering in P. pastoris based on Golden Gate cloning. For this purpose, we established a wide variety of standardized genetic parts (20 promoters of different strength, 10 transcription terminators, 4 genome integration loci, 4 resistance marker cassettes). Results: All genetic parts were characterized based on their expression strength measured by eGFP as reporter in up to four production-relevant conditions. The promoters, which are either constitutive or regulatable, cover a broad range of expression strengths in their active conditions (2-192% of the glyceraldehyde-3-phosphate dehydrogenase promoter PGAP), while all transcription terminators and genome integration loci led to equally high expression strength. These modular genetic parts can be readily combined in versatile order, as exemplified for the simultaneous expression of Cas9 and one or more guide-RNA expression units. Importantly, for constructing multigene constructs (vectors with more than two expression units) it is not only essential to balance the expression of the individual genes, but also to avoid repetitive homologous sequences which were otherwise shown to trigger "loop-out" of vector DNA from the P. pastoris genome. Conclusions: GoldenPiCS, a modular Golden Gate-derived P. pastoris cloning system, is very flexible and efficient and can be used for strain engineering of P. pastoris to accomplish pathway expression, protein production or other applications where the integration of various DNA products is required. It allows for the assembly of up to eight expression units on one plasmid with the ability to use different characterized promoters and terminators for each expression unit. GoldenPiCS vectors are available at Addgene. [ABSTRACT FROM AUTHOR]

The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTauP301L-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.