Selection of Fusion-Site Overhang Sets for High-Fidelity and High-Complexity Golden Gate Assembly.

Golden Gate Assembly depends on the accurate ligation of overhangs at fragment fusion sites to generate full-length products with all parts in the desired order. Traditionally, fusion-site sequences are selected by using validated sets of overhang sequences or by applying a handful of semi-empirical rules to guide overhang choice. While these approaches allow dependable assembly of 6-8 fragments in one pot, recent work has demonstrated that comprehensive measurement of ligase fidelity allows prediction of high-fidelity junction sets that enable much more complex assemblies of 12, 24, or even 36+ fragments in a single reaction that will join with high accuracy and efficiency. In this chapter, we outline the application of a set of online tools that apply these comprehensive datasets to the analysis of existing junction sets, the de novo selection of new high-fidelity overhang sets, the modification and expansion of existing sets, and the principles for dividing known sequences at an arbitrary number of high-fidelity breakpoints.

Modular DNA Construct Design for High-Throughput Golden Gate Assembly.

Golden Gate cloning enables the modular assembly of DNA parts into desired synthetic genetic constructs. The "one-pot" nature of Golden Gate reactions makes them particularly amenable to high-throughput automation, facilitating the generation of thousands of constructs in a massively parallel manner. One potential bottleneck in this process is the design of these constructs. There are multiple parameters that must be considered during the design of an assembly process, and the final design should also be checked and verified before implementation. Doing this by hand for large numbers of constructs is neither practical nor feasible and increases the likelihood of introducing potentially costly errors. In this chapter we describe a design workflow that utilizes bespoke computational tools to automate the key phases of the construct design process and perform sequence editing in batches.

Using ApE for In Silico Golden Gate Cloning.

Golden Gate cloning allows rapid and reliable assembly of multiple DNA fragments in a defined orientation. Golden Gate cloning requires careful design of the restriction fragment overhangs to minimize undesired products and to generate the desired junctions. The ApE (A plasmid Editor) software package can assist in silico design of input fragments or to generate expected assembly products.

Standardized Golden Gate Assembly Metadata Representation Using SBOL.

Synthetic biology, also known as engineering biology, is an interdisciplinary field that applies engineering principles to biological systems. One way to engineer biological systems is by modifying their DNA. A common workflow involves creating new DNA parts through synthesis and then using them in combination with other parts through assembly. Assembly standards such as MoClo, Phytobricks, and Loop are based on Golden Gate, and provide a framework for combining parts. The Synthetic Biology Open Language (SBOL) has implemented a best practice for representing build plans to communicate them to other practitioners through whiteboard designs and in a machine-readable format for communication with lab automation tools. Here we present a software tool for creating SBOL representations of build plans to simulate type IIS-mediated assembly reactions and store relevant metadata.

Automation and Miniaturization of Golden Gate DNA Assembly Reactions Using Acoustic Dispensers.

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.

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in Level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand. Higher order assemblies are typically transcriptional units, and multiple transcriptional units can be assembled into multi-gene constructs. Higher order Golden Gate assembly based on defined and validated parts usually does not introduce sequence changes. Therefore, simple validation of the assemblies, e.g., by colony polymerase chain reaction (PCR) or restriction digest pattern analysis is sufficient. However, in many experimental setups, researchers do not use defined parts, but rather part libraries, resulting in assemblies of high combinatorial complexity where sequencing again becomes mandatory. Here, we present a detailed protocol for the use of a highly multiplexed dual barcode amplicon sequencing using the Nanopore sequencing platform for in-house sequence validation. The workflow, called DuBA.flow, is a start-to-finish procedure that provides all necessary steps from a single colony to the final easy-to-interpret sequencing report.

Use of a Golden Gate Plasmid Set Enabling Scarless MoClo-Compatible Transcription Unit Assembly.

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.

Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

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.

Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning.

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.

Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications.

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.

Construction of Small Genes with Repetitive Elements Using Oligo Extension and Golden Gate Assembly.

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.

Golden Gate Cloning of Expression Plasmids for Synthetic Small RNAs in Bacteria.

Bacterial small RNAs (sRNAs) are well known for their ability to modulate gene expression at the post-transcriptional level. Their rather simple and modular organization provides the user with defined building blocks for synthetic biology approaches. In this chapter, we introduce a plasmid series for Escherichia coli and describe protocols for fast and efficient construction of synthetic sRNA expression plasmids based on Golden Gate assembly. In addition, we present the G-GArden tool, which assists with the design of oligodeoxynucleotides and overhangs for scarless assembly strategies. We propose that the presented procedures are suitable for many applications in different bacteria, which are related to E. coli and beyond.

Golden Gate Cloning of Synthetic CRISPR RNA Spacer Sequences.

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.

Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.

Phage engineering is an emerging technology due to the promising potential application of phages in medical and biotechnological settings. Targeted phage mutagenesis tools are required to customize the phages for a specific application and generate, in addition to that, so-called designer phages. CRISPR-Cas technique is used in various organisms to perform targeted mutagenesis. Yet, its efficacy is notably limited for phage mutagenesis due to the highly abundant phage DNA modifications. Addressing this challenge, we have developed a novel approach that involves the temporal removal of phage DNA cytosine modifications, allowing for effective CRISPR-Cas targeting and subsequent introduction of mutations into the phage genome. The removal of cytosine modification relies on the catalytic activity of a eukaryotic ten-eleven translocation methylcytosine (TET) dioxygenase. TET enzymes iteratively de-modify methylated or hydroxymethylated cytosines on phage DNA. The temporal removal of cytosine modification ultimately enables efficient DNA cleavage by Cas enzymes and facilitates mutagenesis. To streamline the application of the coupled TET-CRISPR-Cas system, we use Golden Gate cloning for fast and efficient assembly of a vector that comprises a TET oxidase and a donor DNA required for scarless site-specific phage mutagenesis. Our approach significantly advances the engineering of modified phage genomes, enabling the efficient generation of customized phages for specific applications.

Golden Gate-Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria.

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria.

Protocol for NT-CRISPR: A Method for Efficient Genome Engineering in Vibrio natriegens.

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.

Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.

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.

Golden Gate Cloning in Actinobacteria: Opportunities and Challenges.

As we exploit biological machineries and circuits to redesign nature, it is just important to use efficient cloning strategies and methods to heterologously express the resulting DNA constructs. Golden Gate cloning allows the assembly of multiple fragments in a single reaction, making the process efficient and seamless. Although Golden Gate strategies have already been employed for different organisms, it is still not well-established for Actinobacteria. Here, we describe methods for Golden Gate cloning and how it can be utilized for Actinobacteria.

Golden Gate Cloning for the Standardized Assembly of Gene Elements with Modular Cloning in Chlamydomonas.

Modern synthetic biology requires fast and efficient cloning strategies for the assembly of new transcription units or entire pathways. Modular Cloning (MoClo) is a standardized synthetic biology workflow, which has tremendously simplified the assembly of genetic elements for transgene expression. MoClo is based on Golden Gate Assembly and allows to combine genetic elements of a library through a hierarchical syntax-driven pipeline. Here we describe the assembly of a genetic cassette for transgene expression in the single-celled model alga Chlamydomonas reinhardtii.

Golden Gate Cloning for Efficient Biosynthesis of Lycopene in Synthetic Yeast.

Golden Gate Assembly (GGA) represents a versatile method for assembling multiple DNA fragments into a single molecule, which is widely used in rapid construction of complex expression cassettes for metabolic engineering. Here we describe the GGA method for facile construction and optimization of lycopene biosynthesis pathway by the combinatorial assembly of different transcriptional units (TUs). Furthermore, we report the method for characterizing and improving lycopene production in the synthetic yeast chassis.