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.

The long journey towards standards for engineering biosystems: Are the Molecular Biology and the Biotech communities ready to standardise?

Synthetic biology needs to adopt sound scientific and industry-like standards in order to achieve its ambitious goals of efficient and accurate engineering of biological systems.

Structural basis for recognition and repair of the 3'-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis.

NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3'-phosphate and other 3'-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3'-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3'-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3'-phosphatase NExo bound to DNA in the presence and absence of a 3'-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3'-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.

One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy.

Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications.

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis.

Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5' to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures.

Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy.

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the "binding" and "base flipping" steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.

Specialization of an Exonuclease III family enzyme in the repair of 3' DNA lesions during base excision repair in the human pathogen Neisseria meningitidis.

We have previously demonstrated that the two Exonuclease III (Xth) family members present within the obligate human pathogen Neisseria meningitidis, NApe and NExo, are important for survival under conditions of oxidative stress. Of these, only NApe possesses AP endonuclease activity, while the primary function of NExo remained unclear. We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo. We demonstrate that the bi-functional meningococcal glycosylases Nth and MutM can perform strand incisions at abasic sites in addition to NApe. However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes. A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level. This specialization of two Xth enzymes for the 3'-end processing and strand-incision reactions has not previously been observed and provides a new paradigm within the prokaryotic world for separation of these critical functions during base excision repair.

A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis.

Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterized meningococcal base excision repair enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However, MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the mismatch repair pathway provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence shows that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes.

Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway.

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.

Revealing the Host-Dependent Nature of an Engineered Genetic Inverter in Concordance with Physiology.

Broad-host-range synthetic biology is an emerging frontier that aims to expand our current engineerable domain of microbial hosts for biodesign applications. As more novel species are brought to "model status," synthetic biologists are discovering that identically engineered genetic circuits can exhibit different performances depending on the organism it operates within, an observation referred to as the "chassis effect." It remains a major challenge to uncover which genome-encoded and biological determinants will underpin chassis effects that govern the performance of engineered genetic devices. In this study, we compared model and novel bacterial hosts to ask whether phylogenomic relatedness or similarity in host physiology is a better predictor of genetic circuit performance. This was accomplished using a comparative framework based on multivariate statistical approaches to systematically demonstrate the chassis effect and characterize the performance dynamics of a genetic inverter circuit operating within 6 Gammaproteobacteria. Our results solidify the notion that genetic devices are strongly impacted by the host context. Furthermore, we formally determined that hosts exhibiting more similar metrics of growth and molecular physiology also exhibit more similar performance of the genetic inverter, indicating that specific bacterial physiology underpins measurable chassis effects. The result of this study contributes to the field of broad-host-range synthetic biology by lending increased predictive power to the implementation of genetic devices in less-established microbial hosts.

Standardization of Fluorescent Reporter Assays in Synthetic Biology across the Visible Light Spectrum.

In synthetic biology, Fluorescent reporters are frequently used to characterize the expression levels obtained from both genetic parts such as promoters and ribosome binding sites as well as from complex genetic circuits. To this end, plate readers offer an easy and high-throughput way of characterizing both the growth and fluorescence expression levels of cell cultures. However, despite the similar mode of action used in different devices, their output is not comparable due to intrinsic differences in their setup. Additionally, the generated output is expressed using arbitrary units, limiting reliable comparison of results to measurements taken within one single experiment using one specific plate reader, hampering the transferability of data across different plate readers and laboratories. This article presents an easy and accessible calibration method for transforming the device-specific output into a standardized output expressing the amount of fluorescence per well as a known equivalent fluorophore concentration per cell for fluorescent reporters spanning the visible light spectrum. This calibration method follows a 2-fold approach determining both the estimated number of cells and the equivalent chemical fluorophore concentration per well. It will contribute to the comparison of plate reader experiments between different laboratories across the world and will therefore greatly improve the reliability and exchange of both results and genetic parts between research groups.

DNA tunneling detector embedded in a nanopore.

We report on the fabrication and characterization of a DNA nanopore detector with integrated tunneling electrodes. Functional tunneling devices were identified by tunneling spectroscopy in different solvents and then used in proof-of-principle experiments demonstrating, for the first time, concurrent tunneling detection and ionic current detection of DNA molecules in a nanopore platform. This is an important step toward ultrafast DNA sequencing by tunneling.

basicsynbio and the BASIC SEVA collection: software and vectors for an established DNA assembly method.

Standardized deoxyribonucleic acid (DNA) assembly methods utilizing modular components provide a powerful framework to explore designs and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use and enables hierarchical assemblies through an idempotent format. These features enable applications including pathway engineering, ribosome binding site (RBS) tuning, fusion protein engineering and multiplexed guide ribonucleic acid (RNA) expression. In this work, we present basicsynbio, open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio), enabling BASIC construct design via simple drag-and-drop operations or programmatically. With basicsynbio, users can access commonly used BASIC parts and linkers while designing new parts and assemblies with exception handling for common errors. Users can export sequence data and create instructions for manual or acoustic liquid-handling platforms. Instruction generation relies on the BasicBuild Open Standard, which is parsed for bespoke workflows and is serializable in JavaScript Object Notation for transfer and storage. We demonstrate basicsynbio, assembling 30 vectors using sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA vector collection is compatible with BASIC and Golden Gate using BsaI. Vectors contain one of six antibiotic resistance markers and five origins of replication from different compatibility groups. The collection is available via Addgene under an OpenMTA agreement. Furthermore, vector sequences are available from within the basicsynbio application programming interface with other collections of parts and linkers, providing a powerful environment for designing assemblies for bioengineering applications. Graphical Abstract.

Synthetic biology and bioelectrochemical tools for electrogenetic system engineering.

Synthetic biology research and its industrial applications rely on deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and electrodes), allowing for the direct integration of electronics with biological processes. However, the use of electrogenetic systems is limited by poor activity, tunability, and standardization. In this work, we developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Last, we demonstrated electrochemical activation of gene expression under aerobic conditions using a novel, modular bioelectrochemical device. These genetic and electrochemical tools facilitate the design and improve the performance of electrogenetic systems. Furthermore, the genetic design strategies used can be applied to other redox-responsive promoters to further expand the available tools for electrogenetics.

The automated Galaxy-SynBioCAD pipeline for synthetic biology design and engineering.

Here we introduce the Galaxy-SynBioCAD portal, a toolshed for synthetic biology, metabolic engineering, and industrial biotechnology. The tools and workflows currently shared on the portal enables one to build libraries of strains producing desired chemical targets covering an end-to-end metabolic pathway design and engineering process from the selection of strains and targets, the design of DNA parts to be assembled, to the generation of scripts driving liquid handlers for plasmid assembly and strain transformations. Standard formats like SBML and SBOL are used throughout to enforce the compatibility of the tools. In a study carried out at four different sites, we illustrate the link between pathway design and engineering with the building of a library of E. coli lycopene-producing strains. We also benchmark our workflows on literature and expert validated pathways. Overall, we find an 83% success rate in retrieving the validated pathways among the top 10 pathways generated by the workflows.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

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.

DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology.

Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process is now receiving serious attention. Automation will enable larger builds using less researcher time, while increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package and demonstrated the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, evaluating the promoter, ribosome binding site and gene order design space for a three-gene operon. All 88 constructs were assembled with high accuracy, at a consumables cost of $1.50-$5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT, making it accessible for most labs and democratizing automated DNA assembly.

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).

Robust estimation of bacterial cell count from optical density.

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals  <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.