The Effect of Ultrasonic Treatment on the Binding of the Inclusion Complex β-Cyclodextrin-peppermint Oil with Cellulose Material

The purpose of the research was to measure the increase in the binding of inclusion complexes β-cyclodextrin-peppermint oil (β-CD_PM) to cellulose in cotton and cotton/polyester material with BTCA as the crosslinking agent by applying an ultrasonic bath at room temperature and a frequency of 80 kHz for 10 min. After sonication, the samples were left in a bath for 24 h after which they were dried, thermocondensed and subjected to a number of wash cycles. The treated samples were analysed with Attenuated total reflection (ATR) units heated up to 300 °C (Golden Gate (FTIR-ATR)) to monitor chemical changes indicative of crosslinking, while physico-chemical changes in the samples were monitored by using Fourier transform infrared spectroscopy (FTIR-ATR). Mechanical properties were measured according to EN ISO 13934-1:1999, and coloristic changes were evaluated by the whiteness degree according to CIE (WCIE) and the yellowing index (YI), while antimicrobial activity was determined according to AATCC TM 147-2016. The results show a physico-chemical modification of the UZV-treated cellulosic material. Moreover, partial antimicrobial efficacy on Gram-negative bacteria was confirmed for treated fabrics.

Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean

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.

GoldBricks: An Improved Cloning Strategy that Combines Features of Golden Gate and Biobricks for Better Efficiency and Usability

ABSTRACT With increasing complexity of expression studies and the repertoire of characterized sequences, combinatorial cloning has become a common necessity. Techniques like Biobricks and Golden Gate aim to standardize and speed up the process of cloning large constructs while enabling sharing of resources. The Biobricks format provides a simplified and flexible approach to endless assembly with a compact library and useful intermediates but is a slow process, joining only two parts in a cycle. Golden Gate improves upon the speed with use of TypeIIS enzymes and joins several parts in a cycle but requires a larger library of parts and logistical inefficiencies scale up significantly in the multigene format. We present here a method that provides improvement over these techniques by combining their features. By using Type IIS enzymes in a format like Biobricks, we have enabled a faster and efficient assembly with reduced scarring, which performs at a similarly fast pace as Golden Gate, but significantly reduces library size and user input. Additionally, this method enables faster assembly of operon-style constructs, a feature requiring extensive workaround in Golden Gate. Our format allows such inclusions resulting in faster and more efficient assembly.

Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCE Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals either constitutively or in response to mercury and arsenic exposure. Beyond the synthesis of novel biosensors, our assembly platform can be adapted for numerous applications, including labelling bacteria for fluorescent microscopy, developing gene expression systems, and modifying bacterial genomes.

A Golden Gate-based Plasmid Library for the Rapid Assembly of Biotin Ligase Constructs for Proximity Labelling

Proximity-labelling has emerged as a powerful tool for the detection of weak and transient interactions between proteins as well as the characterization of subcellular proteomes. One proximity labelling approach makes use of a promiscuous bacterial biotin ligase, termed BioID. Expression of BioID (or of its derivates TurboID and MiniTurbo) fused to a bait protein results in the biotinylation of proximal proteins. These biotinylated proteins can then be isolated by affinity purification using streptavidin-coated beads and identified by mass spectrometry. To facilitate the use of proximity-labelling in plants, we have generated a collection of constructs that can be used for the rapid cloning of TurboID and MiniTurbo fusion proteins using the Golden Gate cloning method. To allow for the use of the constructs in a range of experiments we have designed assembly modules that encode the biotin ligases fused to different linkers as well as different commonly used subcellular localization sequences. We demonstrate the functionality of these vectors through biotinylation assays in tobacco ( Nicotiana benthamiana ) plants .

Construction of multicellular yeast networks using the communication toolkit with variable specificity and attenuation

The key next step in synthetic biology is to extend cellular network engineering to the multicellular level by utilizing cell-cell communication for information processing. To facilitate the implementation of multicellular networks in the most commonly used eukaryotic chassis, Saccharomyces cerevisiae, we developed the yeast communication toolkit (YCTK). This toolkit is based on the fungal mating pathway and contains five pheromone-inducible promoters (response parts), eleven pheromones (α-factors; sender parts), eleven pheromone receptors (Ste2; receiver parts), as well as five Bar1 proteases (suppressor parts). All YCTK parts were thoroughly characterized and are compatible with the commonly used yeast Golden Gate cloning standard. We demonstrated the application of the YCTK by implementing several different logic gate-like population networks. Furthermore, we used this toolkit to investigate the pheromone-receptor promiscuity patterns among different yeast species. This toolkit extends currently available resources for construction of complex multicellular eukaryotic networks with varying degrees of promiscuity and attenuation.

The spatial distribution of the woodland communities and their associated environmental drivers in the Golden Gate Highlands National Park, South Africa

The extreme variability in the topography, altitude and climatic conditions in the temperate Grassland Mountains of Southern Africa is associated with the complex mosaic of grassland communities with pockets of woodland patches. Understanding the relationships between plant communities and environmental parameters is essential in biodiversity conservation, especially for current and future climate change predictions. This article focused on the spatial distribution of woodland communities and their associated environmental drivers in the Golden Gate Highlands (GGHNP) National Park in South Africa. A generalized linear model (GLM) assuming a binomial distribution, was used to determine the optimal environmental variables influencing the spatial distribution of the woodland communities. The Coefficient of Variation (CV) was relatively higher for the topographic ruggedness index (68.78%), topographic roughness index (68.03), aspect (60.04%), coarse fragments (37.46%) and the topographic wetness index (31.33) whereas soil pH, bulk density, sandy and clay contents had relatively less variation (2.39%, 3.23%, 7.56% and 8.46% respectively). In determining the optimal number of environmental variables influencing the spatial distribution of woodland communities, roughness index, topographic wetness index, soil coarse fragments, soil organic carbon, soil cation exchange capacity and remote-sensing based vegetation condition index were significant (p 0.05) and positively correlated with the woodland communities. Soil nitrogen, clay content, soil pH, fire and elevation were also significant but negatively correlated with the woodland communities. The area under the curve (AUC) of the receiver operating characteristics (ROC) was 0.81. This was indicative of a Parsimonious Model with explanatory predictive power for determination of optimal environmental variables in vegetation ecology.Conservation implications: The isolated woodland communities are sources of floristic diversity and important biogeographical links between larger forest areas in the wider Drakensberg region. They provide suitable habitats for a larger number of forest species and harbour some of the endemic tree species of South Africa. They also provide watershed protection and other important ecosystem services. Understanding the drivers influencing the spatial distribution and persistence of these woodland communities is therefore key to conservation planning in the area.