A modular RNA interference system for multiplexed gene regulation.

The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.

Marine Microbial-Derived Resource Exploration: Uncovering the Hidden Potential of Marine Carotenoids.

Microbes in marine ecosystems are known to produce secondary metabolites. One of which are carotenoids, which have numerous industrial applications, hence their demand will continue to grow. This review highlights the recent research on natural carotenoids produced by marine microorganisms. We discuss the most recent screening approaches for discovering carotenoids, using in vitro methods such as culture-dependent and culture-independent screening, as well as in silico methods, using secondary metabolite Biosynthetic Gene Clusters (smBGCs), which involves the use of various rule-based and machine-learning-based bioinformatics tools. Following that, various carotenoids are addressed, along with their biological activities and metabolic processes involved in carotenoids biosynthesis. Finally, we cover the application of carotenoids in health and pharmaceutical industries, current carotenoids production system, and potential use of synthetic biology in carotenoids production.

Biosynthetic gene cluster profiling from North Java Sea Virgibacillus salarius reveals hidden potential metabolites.

Virgibacillus salarius 19.PP.SC1.6 is a coral symbiont isolated from Indonesia's North Java Sea; it has the ability to produce secondary metabolites that provide survival advantages and biological functions, such as ectoine, which is synthesized by an ectoine gene cluster. Apart from being an osmoprotectant for bacteria, ectoine is also known as a chemical chaperone with numerous biological activities such as maintaining protein stability, which makes ectoine in high demand in the market industry and makes it beneficial to investigate V. salarius ectoine. However, there has been no research on genome-based secondary metabolite and ectoine gene cluster characterization from Indonesian marine V. salarius. In this study, we performed a genomic analysis and ectoine identification of V. salarius. A high-quality draft genome with total size of 4.45 Mb and 4426 coding sequence (CDS) was characterized and then mapped into the Cluster of Orthologous Groups (COG) category. The genus Virgibacillus has an "open" pangenome type with total of 18 genomic islands inside the V. salarius 19.PP.SC1.6 genome. There were seven clusters of secondary metabolite-producing genes found, with a total of 80 genes classified as NRPS, PKS (type III), terpenes, and ectoine biosynthetic related genes. The ectoine gene cluster forms one operon consists of ectABC gene with 2190 bp gene cluster length, and is successfully characterized. The presence of ectoine in V. salarius was confirmed using UPLC-MS/MS operated in Multiple Reaction Monitoring (MRM) mode, which indicates that V. salarius has an intact ectoine gene clusters and is capable of producing ectoine as compatible solutes.

Toward Multiplexed Optogenetic Circuits.

Owing to its ubiquity and easy availability in nature, light has been widely employed to control complex cellular behaviors. Light-sensitive proteins are the foundation to such diverse and multilevel adaptive regulations in a large range of organisms. Due to their remarkable properties and potential applications in engineered systems, exploration and engineering of natural light-sensitive proteins have significantly contributed to expand optogenetic toolboxes with tailor-made performances in synthetic genetic circuits. Progressively, more complex systems have been designed in which multiple photoreceptors, each sensing its dedicated wavelength, are combined to simultaneously coordinate cellular responses in a single cell. In this review, we highlight recent works and challenges on multiplexed optogenetic circuits in natural and engineered systems for a dynamic regulation breakthrough in biotechnological applications.

Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs.

Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.

BASIC: A Simple and Accurate Modular DNA Assembly Method.

Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, robust, and highly accurate DNA assembly method, which provides 99% correct assemblies for a typical four-part assembly, enabling high efficiency cloning workflows (Storch et al., ACS Synth Biol, https://doi.org/10.1021/sb500356 , 2015). BASIC employs standardised DNA linkers to combine bioparts, stored in the universal BASIC format. Once a new biopart is formatted into BASIC standard, defined by flanking 18 bp prefix and suffix sequences, it can be placed at any position and in any context within a designed BASIC assembly. This modularity of the BASIC approach is further enhanced by a range of functional linkers, including genetic elements like ribosomal binding sites (RBS) and peptide linkers. The method has a single tier format, whereby any BASIC assembly can create a new composite BASIC part in the same format used for the original parts; it can thus enter a subsequent BASIC assembly without the need for reformatting or changes to the workflow. This unique idempotent cloning mechanism allows for the assembly of constructs in multiple, conceptionally simple hierarchical rounds. Combined with its high accuracy and robustness, this makes BASIC a versatile assembly method for combinatorial and complex assemblies both at bench and biofoundry scale. The single universal storage format of BASIC parts enables compressed universal biopart libraries that promote sharing of parts and reproducible assembly strategies across labs, supporting efforts to improve reproducibility. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol, relies on a single tier format, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round (Casini et al., Nat Rev Mol Cell Biol. https://doi.org/10.1038/nrm4014 , 2015).

Quantification of bacterial fluorescence using independent calibrants.

Fluorescent reporters are commonly used to quantify activities or properties of both natural and engineered cells. Fluorescence is still typically reported only in arbitrary or normalized units, however, rather than in units defined using an independent calibrant, which is problematic for scientific reproducibility and even more so when it comes to effective engineering. In this paper, we report an interlaboratory study showing that simple, low-cost unit calibration protocols can remedy this situation, producing comparable units and dramatic improvements in precision over both arbitrary and normalized units. Participants at 92 institutions around the world measured fluorescence from E. coli transformed with three engineered test plasmids, plus positive and negative controls, using simple, low-cost unit calibration protocols designed for use with a plate reader and/or flow cytometer. In addition to providing comparable units, use of an independent calibrant allows quantitative use of positive and negative controls to identify likely instances of protocol failure. The use of independent calibrants thus allows order of magnitude improvements in precision, narrowing the 95% confidence interval of measurements in our study up to 600-fold compared to normalized units.