Golden Gate Research Articles

In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly

Abstract Golden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation. Most Golden Gate strategies rely on four nucleotide overhangs generated by commonly used Type IIS enzymes. However, reduction to three nucleotide overhangs allows the use of codons as fusion sites and reduces potential scar sequences. This is particularly important when studying biological functions, as additional nucleotides may alter the structure or stability of the transcribed RNA. To address this issue we use SapI, a Type IIS enzyme generating three nucleotide overhangs, for transcription unit assembly, allowing for codon-based fusion in coding sequences. We created a corresponding plasmid toolbox for basic part generation and transcription unit assembly, a workflow we term In-Cloning. In-Cloning is downstream compatible with the Modular Cloning standard developed by Sylvestre Marillonnet’s group for standardized assembly of multigene constructs. However, the multigene construct plasmids may not be compatible for use with the model organism of choice. Therefore, we have developed a workflow called Out-Cloning to rapidly generate Golden Gate acceptor plasmids. Out-Cloning uses standardized plasmid parts that are assembled into Golden Gate acceptor plasmids using flexible linkers. This allows the systematic construction of acceptor plasmids needed to transfer assembled DNA into the organism of interest.

Brushes with History

Against a backdrop of other murals marking national territories and Manifest Destiny—notably, William Leutze’s stereochrome Westward the Course of Empire Takes Its Way completed for the U.S. Capitol during the Civil War, with its iconic depiction of a mostly monochrome van of westward migrants on their way over mountains to California’s Golden Gate—this article focuses on the way artists Judy Baca and Ana Teresa Fernández have both deconstructed physical and symbolic walls to open up a view of migrations across the U.S.–Mexico border as natural. The essay examines how each artist’s work gets to the same end vision but through different starting points in relation to history. Baca—in her ongoing History of California (a.k.a., the Great Wall of Los Angeles), and in her mural La Memoria de Nuestra Tierra, California—has brought repressed or erased history and people to light, and she explicitly envisions the border wall as unnatural. By contrast, Fernández optically erases the border wall at Tijuana / San Ysidro / the Pacific and other places along la frontera, employing Martha Stewart paint to create chinks and gashes in the wall’s ideological veneer, brushing blue onto corroding iron to, she says, pull “down the sky to kiss the ground again.” Baca represents what has been erased while Fernández erases what has been represented, but both do so to reconnect a California landscape that has been bifurcated by the border wall. Considered in relation to the history and historiography of the U.S.–Mexico borderlands—and in particular to work on the physical manifestation of the border through markers, monuments, fences and walls designed to split lands and peoples—this article considers these artists’ work as powerful demurrals, defiant visual protests and imaginaries that affirm inclusion, empathy, and human and ecological connections across the border between Alta and Baja California.

Two Verse Novels — Two “Onegins”: Interlingual and Intercultural Translation

This article explores the connection between two literary works: Alexander Pushkin’s “Eugene Onegin” and Vikram Seth’s verse novel “The Golden Gate,” written more than 150 years after Pushkin’s text and referred to by critics as the “Eugene Onegin of San Francisco.” The aim of this study is a comparative analysis of the two novels, high-lighting Pushkin’s influence on the Anglophone text by the Indian author. Through the analysis, it is concluded that there are numerous borrowings present in Seth’s novel, including the Onegin stanza, the formula for introducing the main character, and various allusions and direct references to Pushkin’s text. The research material also encompasses the English translation of “Eugene Onegin” by Charles Johnston, which influenced Seth. The novelty of this research lies in the consideration of “The Golden Gate” as a form of double translation: both interlingual and intercultural. It is demonstrated that Seth’s work emerged as a result of the English translation of Pushkin’s verse novel, which served as a pretext that he reinterpreted within the context of twentieth-century American culture. This double translation unites two authors, two cultures, and two epochs.

Golden EGG, a simplified Golden Gate cloning system to assemble multiple fragments

The Golden Gate method is an efficient tool for seamless assembly of multiple DNA fragments, which uses Type IIS restriction endonucleases, cleaving the DNA outside of their recognition site to release DNA parts from PCR fragments or entry clones, thus allowing the design of overhangs for ligation at will. However, the construction of the entry clones requires the use of other restriction enzyme(s) or cloning techniques and different entry vectors for the individual overhangs. Here, we present a simplified Golden Gate cloning approach termed Golden EGG. It features (1) a single entry vector with a specific cloning site to host the DNA parts; (2) a unique primer design to create the restriction enzyme recognition site to release the fragments with the overhangs at will; (3) the use of a single Type IIS enzyme for the construction of both the entry and destination clones; (4) a specific temperature profile during the digestion-ligation reaction. Our user-friendly, streamlined method retains the key attributes of the Golden Gate technique, while offering the potential to generate compatible parts with any existing Golden Gate toolkit and to be accessible to a wide user base without the need for extensive acquisition of new vectors or expensive enzymes.

Surface display and application of cadmium-binding protein CADR on the cell wall of Chlamydomonas reinhardtii

Algae has been proven to have the potential to be efficient biosorbents in the detection and remediation of heavy metal pollution such as cadmium in the environment. This study aims to enhance the cadmium adsorption capacity of Chlamydomonas reinhardtii by expressing the cadmium-binding protein CADR on the cell wall by the surface display technology. Firstly, the golden gate technique was employed to construct the transformation vector PET-X-CADR, which anchored CADR to the cell wall with the cell wall protein GP1. The high-throughput screening with the fluorescence signal of the fusion tag YFP resulted in three engineered algal strains with high expression of CADR on the cell wall. Physiological experiments demonstrated that the CADR displayed on the cell wall did not affect the growth of the engineered algal strains exposed to cadmium with the concentration below 200 μmol/L. In the presence of 200 μmol/L cadmium, the growth rates of CADR-engineered algal strains were three times as that of the wild-type, indicating stronger tolerance of the CADR-engineered algae to cadmium. The protein lyase GLE released during the mating of Chlamydomonas was used to isolate the cell walls of wild-type and engineered strains, the cadmium content of which was compared. The results showed that the cell wall of the engineered strain exhibited an increase of 33% in cadmium adsorption capacity. This study gives insights into the application of algae in the management of cadmium pollution in the environment, especially in the recycling of heavy metals from the environment.

Improved gene editing and fluorescent-protein tagging in Aspergillus nidulans using a Golden Gate-based CRISPR-Cas9 plasmid system

CRISPR-Cas9 systems can be used for precise genome editing in filamentous fungi, including Aspergillus nidulans. However, current CRISPR-Cas9 systems for A. nidulans rely on relatively complex or multi-step cloning methods to build a plasmid expressing both Cas9 and an sgRNA targeting a genomic locus. In this study we improve on existing plasmid-based CRISPR-Cas9 systems for Aspergilli by creating an extremely simple-to-use CRISPR-Cas9 system for A. nidulans genome editing. In our system, a plasmid containing both Cas9 and an sgRNA is assembled in a one-step Golden Gate reaction. We demonstrate precise, scarless genome editing with nucleotide-level DNA substitutions, and we demonstrate markerless gene tagging by fusing fluorescent-protein coding sequences to the endogenous coding sequences of several A. nidulans genes. We also describe A. nidulans codon-adjusted versions of multiple recent-generation fluorescent proteins, which will be useful to the wider Aspergillus community.

Golden gate method in biology and medicine

The aim of this review was to describe and compare molecular cloning methods for assembling genetic constructs. Genetic therapy is one of the rapidly developing fields of modern medicine, so special attention in this review is paid to parameters such as speed, accuracy, and efficiency of cloning, as these are critical factors in creating gene therapy agents. Special attention is given to the Golden Gate method, which is based on the use of type IIS restriction endonucleases, as this approach simplifies the cloning process and increases its efficiency through standardized design and a minimal set of enzymes. Alongside Golden Gate, the review also discusses more innovative molecular cloning methods, Gateway and Gibson, in terms of their potential use for addressing fundamental and applied challenges in regenerative medicine.

Enhanced Golden Gate Assembly: evaluating overhang strength for improved ligation efficiency.

Molecular cloning, a routine yet essential technique, relies heavily on efficient ligation, which can be significantly improved using Golden GateAssembly (GGA). A key component of GGA is the use of type IIS enzymes, which uniquely cleave downstream of their recognition sequences to generate various overhangs, including non-palindromic ones. Recent advancements in GGA include the development of newly engineered enzymes with enhanced activity. Additionally, high-throughput GGA assays, which allow for the simultaneous study of all possible overhangs, have identified optimal GGA substrates with high efficiencies and fidelities, greatly facilitating the design of complex assemblies. Interestingly, these assays reveal unexpected correlations between ligation efficiencies and overhang stabilities. One hypothesis for this observation is that newly hydrolyzed DNA fragments with strong overhangs can readily re-ligate, thereby slowing down the overall process. In this paper, we employ a combination of gel electrophoresis and numerical calculations to test this hypothesis, ultimately determining that it does not hold true under the conditions established by conventional GGA assays. Using an assembly of 10 fragments, we demonstrate that strong overhangs yield higher GGA efficiency, while weak overhangs result in lower efficiency. These findings enable us to propose optimal overhangs for efficient GGA assays, significantly increasing yield.

GGAssembler: Precise and economical design and synthesis of combinatorial mutation libraries.

Golden Gate assembly (GGA) can seamlessly generate full-length genes from DNA fragments. In principle, GGA could be used to design combinatorial mutation libraries for protein engineering, but creating accurate, complex, and cost-effective libraries has been challenging. We present GGAssembler, a graph-theoretical method for economical design of DNA fragments that assemble a combinatorial library that encodes any desired diversity. We used GGAssembler for one-pot in vitro assembly of camelid antibody libraries comprising >105 variants with DNA costs <0.007$ per variant and dropping significantly with increased library complexity. >93% of the desired variants were present in the assembly product and >99% were represented within the expected order of magnitude as verified by deep sequencing. The GGAssembler workflow is, therefore, an accurate approach for generating complex variant libraries that may drastically reduce costs and accelerate discovery and optimization of antibodies, enzymes and other proteins. The workflow is accessible through a Google Colab notebook at https://github.com/Fleishman-Lab/GGAssembler.

Development of a Golden Gate Assembly-Based Genetic Toolbox for Lactiplantibacillus plantarum and Its Application for Engineering Monoterpenoid Biosynthesis.

Lactiplantibacillus plantarum is a food-grade lactic acid bacterium widely used in the food and beverage industry. Recently, this probiotic organism has been applied as a biofactory for the production of pharmaceutical and food-related compounds, but existing promoters and expression vectors for the genetic engineering of L. plantarum rely on inefficient cloning strategies and are usually not well-characterized. We therefore developed a modular and standardized Golden Gate Assembly-based toolbox for the de novo assembly of shuttle vectors from Escherichia coli to L. plantarum. A collection of the most relevant genetic parts, e.g., different origins of replication and promoters, was incorporated in our toolbox and thoroughly characterized by flow cytometry and the fluorescence assay. Standardized fusion sites allow combining the genetic part freely into a plasmid in one step. This approach allows for the high-throughput assembly of numerous constructs in a standardized genetic context, thus improving the efficiency and predictability of metabolic engineering in L. plantarum. Using our toolbox, we were able to produce the aroma compounds linalool and geraniol in L. plantarum by extending its native mevalonate pathway with plant-derived monoterpenoid synthases.

Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly

The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

Golden Gate: Building the Mighty Bridge by Elizabeth Partridge (review)

Golden Gate: Building the Mighty Bridge by Elizabeth Partridge (review)

Multiplex Expression Cassette Assembly: A flexible and versatile method for building complex genetic circuits in conventional vectors.

The manipulation of multiple transcription units for simultaneous and coordinated expression is not only key to building complex genetic circuits to accomplish diverse functions in synthetic biology, but is also important in crop breeding for significantly improved productivity and overall performance. However, building constructs with multiple independent transcription units for fine-tuned and coordinated regulation is complicated and time-consuming. Here, we introduce the Multiplex Expression Cassette Assembly (MECA) method, which modifies canonical vectors compatible with Golden Gate Assembly, and then uses them to produce multi-cassette constructs. By embedding the junction syntax in primers that are used to amplify functional elements, MECA is able to make complex constructs using only one intermediate vector and one destination vector via two rounds of one-pot Golden Gate assembly reactions, without the need for dedicated vectors and a coherent library of standardized modules. As a proof-of-concept, we modified eukaryotic and prokaryotic expression vectors to generate constructs for transient expression of green fluorescent protein and β-glucuronidase in Nicotiana benthamiana, genome editing to block monoterpene metabolism in tomato glandular trichomes, production of betanin in tobacco and synthesis of β-carotene in Escherichia coli. Additionally, we engineered the stable production of thymol and carvacrol, bioactive compounds from Lamiaceae family plants, in glandular trichomes of tobacco. These results demonstrate that MECA is a flexible, efficient and versatile method for building complex genetic circuits, which will not only play a critical role in plant synthetic biology, but also facilitate improving agronomic traits and pyramiding traits for the development of next-generation elite crops.

From Lahore to New York: Changing Identity of Lahure in Modern Nepali Literature

This article assesses the changing identity of lahure in modern Nepali literature. Beginning with the etymology ‘lahure’ that has its one-to-one correspondence with Lahore in the then British India, this article attempts to map the expansion of that particular identity right from Nepali youths’ entrance to Lahore to serve as a mercenary for Ranjit Singh, the Khalsa warrior king and interestingly not for the British colonials, as early as 1809, that is some time before the Treaty of Sugauli in 1816 that officially opened the gate for Nepali youths to the Company Army. Deriving impetus from Craig Calhoun’s discourse of identity in reading Tulachan Ale’s Manipurko Ladai ko Sawai as the early record of the formation of the lahure identity and Sirjana Sharma’s Golden Gate as the contemporary record of such identity, this article analyzes how the identity of a lahure has significantly changed from the early Nepali youths, that too exclusively males, who visited Lahore as the pioneer migrants to British India to the Nepalis who migrate to the United States after winning lottery via the Diversity Visa Program. This article scrutinizes the loose definition of lahure in Nepali society that not only transcends the etymological meaning of the term but also deconstructs the notion of stereotypical adherence to a specific gender, that is, male.

The Chabahar Transit Project: Transition from Geo-Politics to Geo-Economics

ABSTRACT The Chabahar Transit Project is a multimodal network linking India, Iran, Afghanistan, and, recently, Uzbekistan, through maritime, rail, and road routes. The project results from a complex combination of interests and goals of the member countries and is a clear example of the transition from geo-politics to geo-economics. Iran looks at the Chabahar part as the only ocean port of the cautery and golden gate to landlocked Afghanistan and Central Asia to increase Iran’s capacity in the International North-South Transport Corridor (INSTC). India looks at the Chabahar transit project as a part of the “Diamond Necklace Strategy” against China’s “String of Pearls” Strategy and also bypassing the traditional rival, Pakistan, especially to counter China’s massive investment in the development of Pakistan’s Gwadar port, as well as the China-Pakistan Economic Corridor (CPEC). Finally, the Russo-Ukraine war and the block of east-west transit corridors traversing Russian territory into Europe have increased the role of the Chabahar transit project in the INSTC. However, the possible failure of negotiations to revive the JCPOA and the re-imposition of UN Chapter 7 sanctions against Iran is the most crucial challenge for the Chabahar transit project and the India-Iran-Afghanistan-Uzbekistan quadrilateral agreement.

Redesigning the Drosophila histone gene cluster: an improved genetic platform for spatiotemporal manipulation of histone function.

Mutating replication-dependent (RD) histone genes is an important tool for understanding chromatin-based epigenetic regulation. Deploying this tool in metazoans is particularly challenging because RD histones in these organisms are typically encoded by many genes, often located at multiple loci. Such gene arrangements make the ability to generate homogenous histone mutant genotypes by site-specific gene editing quite difficult. Drosophila melanogaster provides a solution to this problem because the RD histone genes are organized into a single large tandem array that can be deleted and replaced with transgenes containing mutant histone genes. In the last ∼15 years several different RD histone gene replacement platforms were developed using this simple strategy. However, each platform contains weaknesses that preclude full use of the powerful developmental genetic capabilities available to Drosophila researchers. Here we describe the development of a newly engineered platform that rectifies many of these weaknesses. We used CRISPR to precisely delete the RD histone gene array (HisC), replacing it with a multifunctional cassette that permits site-specific insertion of either one or two synthetic gene arrays using selectable markers. We designed this cassette with the ability to selectively delete each of the integrated gene arrays in specific tissues using site-specific recombinases. We also present a method for rapidly synthesizing histone gene arrays of any genotype using Golden Gate cloning technologies. These improvements facilitate the generation of histone mutant cells in various tissues at different stages of Drosophila development and provide an opportunity to apply forward genetic strategies to interrogate chromatin structure and gene regulation.

Exploring environmental factors that influence the distribution of poplar trees

AbstractPoplar is an invasive alien plant (IAP) that encroaches on fragile landscapes in South Africa, but the environmental factors that contribute to its spread are not well understood. To address this gap, we conducted a study using generalised linear models (GLM) to identify the most significant local environmental factors that drive poplar establishment and dispersion in Golden Gate Highlands National Park (GGHNP). We then ranked these variables according to their contribution to poplar occurrence using a random forests regression approach. Our GLM analysis revealed that soil properties such as soil organic carbon, sand and silt, topographical variables such as aspect and slope, normalised difference moisture index (NDMI) and poplar distance from rivers are significant factors affecting poplar survival. After ranking the variables, we found that the three most important factors influencing poplar occurrence were aspect, slope and poplar distance to rivers, followed by the sand, silt, NDMI, and soil organic carbon. Our results are consistent with previous studies, indicating that topographical variables and the availability of sufficient water supply are the primary drivers of the spread of IAPs. This study provides useful insights in managing and monitoring poplars in delicate landscapes.

Grass curing-driven fire danger index in a protected mountainous grassland using fused MODIS and Sentinel-2

ABSTRACT This study assessed the spatio-temporal variation of the degree of curing (DoC) in the Golden Gate Highlands National Park (GGHNP) from 2016 to 2020. The estimation of the degree of grass curing was carried out with the high spatial resolution fused remotely sensed data of MODIS and Sentinel-2 employing Index-then-Blend (IB) data fusion methods on Google Earth Engine (GEE) to compute Grassland Curing Index (GCI) using soil and vegetation moisture content indices-based grassland curing algorithm. Grassland curing indices were computed on Google Earth Engine and converted into Grassland Curing Maps (GCMs), which were then synthesized into fire danger maps. The study showed that all selected indices have the highest DoC in the month of September, with the minor time variation observed in MODIS and Sentinel 2-derived GCI estimated using Shortwave Infrared Water Stress (GCI_SIWSI) in the months of August and March, respectively. The analysis further revealed that GCI_SIWSI (44%) derived from Sentinel-2 data yielded the highest area of extremely high fire danger, followed by GCI estimated using Normalized Difference Moisture Index (GCI_NDMI) (25%) derived from fused data, MODIS-derived GCI_SIWSI and fused-derived GCI estimated using Global Vegetation Monitoring Index (GCI_GVMI) (both 16%). The GCI_SIWSI derived from Sentinel-2 data also showed that more than 95% of the entire landmass of the study area was within a high to extremely high fire danger zone, making the park’s vegetation susceptible to fire. Among all the four selected indices correlated against fire point, only SIWSI revealed a positive relationship across MODIS, Sentinel-2 and Fused data. It is also evident from the study that GCMs derived from Fused data outperformed MODIS and Sentinel-2 with the highest R2 and F values of 0.65 and 380, respectively. These results indicate that GCI derived from fused remotely sensed data is a promising GCI for accurately assessing the DoC over mountainous grassland environments. The study paved the way for increasing the spatial resolution for estimating the DoC for fire and fuel management of parks and other fire agencies within mountainous grassland environments.

Robust, high-yield, rapid fabrication of DNA constructs for Magnetic Tweezers

Single-molecule techniques are highly sensitive tools that can reveal reaction intermediates often obscured in experiments involving large ensembles of molecules. Therefore, they provide unprecedented information on the mechanisms that control biomolecular reactions. Currently, one of the most significant single-molecule assays is Magnetic Tweezers (MT), which probes enzymatic reactions at high spatio-temporal resolutions on tens, if not hundreds, of molecules simultaneously. For high-resolution MT experiments, a short double-stranded DNA molecule (less than 2000 base pairs) is typically attached between a micron-sized superparamagnetic bead and a surface. The fabrication of such a substrate is key for successful single-molecule assays, and several papers have discussed the possibility of improving the fabrication of short DNA constructs. However, reported yields are usually low and require additional time-consuming purification steps (e.g., gel purification). In this paper, we propose the use of a Golden Gate Assembly assay that allows for the production of DNA constructs within minutes (starting from PCR products). We discuss how relevant parameters may affect the yield and offer single-molecule experimentalists a simple yet robust approach to fabricate DNA constructs.

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.