IFLinkC-EZ: A scalable and automatable method for the assembly of complex fusion proteins and multi-gene expression constructs based on the iFLinkC framework.

An introduction to synthetic biology in plant systems: ERASynBio/OpenPlant summer school for early career researchers, September 2014.

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.

<p indent=0mm>Synthetic biology applies engineering principles for the deliberate design, engineering, and <italic>de novo</italic> creation of artificial biological systems with certain functions. Due to the complexity of living systems and lack of rational design principles, iterative trial-and-error experiments are often necessary, but the dependence on human researchers limits the throughput, efficiency, and consistency of such endeavor. To overcome these limitations, biofoundries are developed as an integrated infrastructure for accelerating the “design-build-test-learn” cycles in synthetic biology research and biotechnology applications. Computer-aided design and robotic automation are applied in the physical manufacturing and prototyping of engineered DNA and genetically reprogrammed organisms. Currently, many biofoundries are being created around the world, and a Global Biofoundry Alliance has been established to promote coordination and collaboration. This paper aims to summarize the recent advances in synthetic biology automation and introduce design and construction of current and future biofoundries. We start with key technologies that promote automation in synthetic biology, including computer-aided design, high-throughput instrumentation, robotic integration, automation-compatible workflows for DNA assembly and chassis engineering, and analytical approaches. For bio-design automation, we introduce existing software tools such as j5 from Agile Biofoundry, iBioCAD from Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB), and CUBA from Edinburgh Genome Foundry (EGF). Moreover, software and hardware for creating automated build and test workflows are summarized, followed by recent advances in DNA synthesis, DNA assembly, nucleic acid extraction and analysis, chassis manipulation, and high-throughput testing. Notable examples are discussed, including automated DNA assembly using the Golden-Gate method, multiplex automated yeast genome engineering, and artificial intelligence (AI)-guided optimization of biosynthetic pathways. Then, representative biofoundries and their software systems, robotic platforms, and available workflows are discussed. Biofoundries are categorized by various formats of integration, including full integration (e.g., iBioFAB and EGF), modular integration (e.g., London Biofoundry, Singapore Biofoundry, and Concordia Genome Foundry), and manual integration. Commercial biofoundries are also compared and contrasted with their academic counterparts, using the ones at Gingko Bioworks Inc. as an example. Furthermore, the design of Shenzhen Biofoundry is discussed in detail, whereby a centralized cloud lab is envisioned to serve the domestic and international synthetic biology communities. Shenzhen Biofoundry will consist of a build platform for engineered DNA, phage, bacteria and yeast, a test platform for optics, chromatography and mass spectrometry, and fermentation scale-up, and a design/learn/cloud platform to coordinate and integrate the whole facility. We conclude with the future challenges and promises of biofoundries and synthetic biology automation. We propose key research directions, including new automation technologies, data-driven and intelligent design, and automation-compatible workflows in synthetic biology. Systems approach and interdisciplinary collaboration are necessary for biofoundries development, which requires synergistic integration of synthetic biology, analytical chemistry, robotics, instrumentation, informatics, and smart manufacturing. Just like foundries initiated the magnificent prosperity of the semiconductor industry, biofoundries will unleash the potential of synthetic biology to revolutionize our society.

The localization in space and in time of proteins within the cytoplasm of eukaryotic cells is a central question of the cellular compartmentalization of metabolic pathways. The assembly of proteins within stable or transient complexes plays an essential role in this process. Here, we examined the subcellular localization of the multi-aminoacyl-tRNA synthetase complex in human cells. The sequestration of its components within the cytoplasm rests on the presence of the eukaryotic-specific polypeptide extensions that characterize the human enzymes, as compared with their prokaryotic counterparts. The cellular mobility of several synthetases, assessed by measuring fluorescence recovery after photobleaching, suggested that they are not freely diffusible within the cytoplasm. Several of these enzymes, isolated by tandem affinity purification, were copurified with ribosomal proteins and actin. The capacity of aminoacyl-tRNA synthetases to interact with polyribosomes and with the actin cytoskeleton impacts their subcellular localization and mobility. Our observations have conceptual implications for understanding how translation machinery is organized in vivo.

Future making and responsible governance of innovation in synthetic biology

β-Catenin Interacts With the FUS Proto-oncogene Product and Regulates Pre-mRNA Splicing

In 2015, the first-ever Slovenian high school team competed at the iGEM (international Genetically Engineered Machine) competition in synthetic biology. The team was carefully selected from a list of candidates proposed by their high school teachers of chemistry and biology. Composed of eight students from 7 high schools from across the country, the team split into two groups, working in two institutions but with regular common meetings. The group of four students who worked at the National Institute of Chemistry focused on biotechnological production and the second group was preparing genetic constructs at the University of Ljubljana Faculty of Chemistry and Chemical Technology. The central problem that students tangled was the conversion of C-4 acids that are side-products of anaerobic waste degradation, into the corresponding alcohol. In the biotechnological part of the project students tested butanoic acid production in an anaerobic fermentation broth using a 15-channel computer-controlled bioreactor system, configured to allow online analysis of gaseous products (by GC) and frequent analysis of dissolved compounds (by HPLC). Next, at optimal butanoic acid production conditions (c = 1 g/L butanoic acid) growth of E. coli was examined in terms of medium composition, temperature and pH in order to obtain optimal operational parameters for biotechnological transformation of butanoic acid to butanol. Overall, E. coli growth was analysed in 72 different growth media in microtiter plates. Students followed inhibition of E. coli growth by butanol, acetone and isobutanol in the presence of butanoic acid and glycerol. The synthetic biology group started with PCR amplification of three genes ( ctf A and ctf B encoding two polypeptide chains of Co-A transferase, and bdh B encoding butyraldehide dehydrogenase for a two.stage conversion of butyril CoA to butanol) from anaerobic microbial community. Amplification failed, probably due to contaminants in the broth and a low number of bacteria harbouring these genes. Consequently, synthetic genes were designed with appended sequences for easier cloning, for a hexahistidine tag for detection of recombinant proteins using specific antibodies. Synthetic genes were inserted into pSB1C3 vectors and for each of them a strong promoter- ribosome binding site region was inserted in front of the enzyme coding region. Such combined constructs were further transferred into vectors with double terminators of transcription. In total, 9 different DNA constructs were prepared and deposited in the Registry of biological parts. All final expression constructs efficiently directed production of recombinant enzymes in E. coli DH5α under aerobic conditions. The giant jamboree of competing teams took place in Boston, USA, from September 24 to 28 with more than 260 teams from around the globe. In the high school track the Slovenian team presented the project entitled “From waste to fuel: Reprogrammed E. coli for sustainable production of biobutanol from butanoic acid”. The project received high recognition, as it was awarded a gold medal for completed tasks and was nominated among five best teams in categories Best Integrated Human Practices, Best Wiki, Best Presentation and Best High School Project.

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.

Temperature-driven differential gene expression by RNA thermosensors

Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one-pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline. © 2020 The Authors. Basic Protocol 1: Performing a typical Golden Gate cloning reaction Basic Protocol 2: Accommodating a vector to Golden Gate cloning Basic Protocol 3: Accommodating an insert to Golden Gate cloning Basic Protocol 4: Generating small standardized parts compatible with hierarchical modular cloning (MoClo) using level 0 vectors Alternate Protocol: Generating large standardized parts compatible with hierarchical modular cloning (MoClo) using level -1 vectors Basic Protocol 5: Assembling transcription units and multigene constructs using level 1, M, and P MoClo vectors.

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.

SummaryFunctional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time‐consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence‐guided nicking endonuclease (UNiE)‐mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15‐nt 3′ single‐strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination‐mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII‐UNiE. Using TGSII‐UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII‐UNiE. In conclusion, UNiEDA is an efficient, convenient and low‐cost method for DNA cloning and multigene stacking, and the TGSII‐UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

Golden Gate cloning has become a powerful and widely used DNA assembly method. Its modular nature and the reusability of standardized parts allow rapid construction of transcription units and multi-gene constructs. Importantly, its modular structure makes it compatible with laboratory automation, allowing for systematic and highly complex DNA assembly. Golden Gate cloning relies on type IIS enzymes that cleave an adjacent undefined sequence motif at a defined distance from the directed enzyme recognition motif. This feature has been used to define hierarchical Golden Gate assembly standards with defined overhangs ("fusion sites") for defined part libraries. The simplest Golden Gate standard would consist of three-part libraries, namely promoter, coding and terminator sequences, respectively. Each library would have defined fusion sites, allowing a hierarchical Golden Gate assembly to generate transcription units. Typically, type IIS enzymes are used, which generate four nucleotide overhangs. This results in small scar sequences in hierarchical DNA assemblies, which can affect the functionality of transcription units. However, there are enzymes that generate three nucleotide overhangs, such as SapI. Here we provide a step-by-step protocol on how to use SapI to assemble transcription units using the start and stop codon for scarless transcription unit assembly. The protocol also provides guidance on how to perform multi-gene Golden Gate assemblies with the resulting transcription units using the Modular Cloning standard. The transcription units expressing fluorophores are used as an example.

This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding.

Voltage Across the Target Cell Membrane is a Strong Regulator of Fusion of Virus Containing Class II or Class III Proteins