Significance Nonmodel bacteria have essential roles to play in the future development of biotechnology by providing new sources biocatalysts, antibiotics, hosts for bioproduction, and engineered “living therapies.” The characterization such can be challenging, as many are not tractable standard molecular biology techniques. This paper presents a rapid automated methodology characterizing DNA parts from nonmodel bacterium using cell-free transcription–translation. Data analysis was performed...

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved subtilis...

Overlap-directed DNA assembly methods allow multiple parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of parts, have become widely adopted synthetic biology, despite being incompatible with a key principle engineering: modularity. To answer this, we present MODAL: Modular Overlap-Directed Assembly Linkers strategy that brings modularity overlap-directed allowing an initial set into variety arrangements one-pot reactions. MODAL is...

Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for biosynthesis numerous different types plant products organized as metabolic gene clusters, thereby unveiling highly unusual form genome architecture and offering novel avenues discovery exploitation specialized metabolism. Here we show these clustered pathways characterized by distinct chromatin signatures histone 3 lysine trimethylation...

R2oDNA Designer is a web application that stochastically generates orthogonal sets of synthetic DNA sequences designed to be biologically neutral. Biologically neutral may used for directing efficient assembly by overlap-directed methods, as negative control functional DNA, barcodes, or potentially spacer regions insulate biological parts from local context. The software creates optimized using Monte Carlo simulated annealing approach followed the elimination homologous host genomes and...

Coarse-grained protein structure models offer increased efficiency in structural modeling, but these must be coupled with fast and accurate methods to revert a full-atom structure. Here, we present novel algorithm reconstruct mainchain from C traces. This has been parameterized by fitting Gaussian mixture (GMMs) short backbone fragments centered on idealized peptide bonds. The method have developed is statistically significantly more than several competing methods, both terms of RMSD values...

The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large gene circuits are increasingly accessible. However, cell engineering, traditional tissue culture methods slow cumbersome, not suited high-throughput characterization measurements. Here we have utilized cell-free protein synthesis (CFPS) assays using HeLa extracts liquid handling automation as an alternative to flow cytometry-based Our CFPS take a few hours, established optimized protocols...

Abstract Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature parasitic is the release specific proteases by at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif...

Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA TssC/VipB, assemble form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages pyocins. Here, we used cryoelectron microscopy solve the structure H1-T6SS P. TssB1C1 sheath at 3.3 Å resolution. Our allowed us resolve some features were not resolved in Vibrio cholerae VipAB Francisella...

Abstract In recent years, there have been significant advances in the field of computational protein design including successful enzymes based on backbone scaffolds from experimentally solved structures. It is likely that large‐scale sampling conformations will become necessary as further progress made more complicated systems. Removing constraint having to use known backbones a potential method solving problem. With this application mind, we describe systematically construct large number de...

Significance The development of algorithms to design new proteins with backbone plasticity is a key challenge in computational protein design. In this paper, we describe class extensible synthetic repeat scaffolds computationally designed variable loops projecting from the central core. We have developed methods sample conformations using coarse-grained potential energy function without fragments known structures. This procedure was combined existing for sequence successfully loop at atomic...

Abstract The thermodynamic and kinetic stabilities of the eye lens family βγ‐crystallins are important factors in etiology senile cataract. They control chance proteins unfolding, which can lead to aggregation loss transparency. βB2‐Crystallin orthologs low stability comprise two typical βγ‐crystallin domains, although, uniquely, N‐terminal domain has a cysteine one conserved folded β‐hairpins. Using high‐temperature (500 K) molecular dynamics simulations with explicit solvent on rodent...

Base excision repair (BER) is a highly conserved DNA pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required complete the multistep process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling by recognizing intermediary abasic sites cleaving phosphodiester backbone 5′ site. Despite extensive study, there no structure bacterial AP endonuclease bound substrate DNA. Furthermore, structural mechanism for AP-site...

Coarse-grained (CG) methods for sampling protein conformational space have the potential to increase computational efficiency by reducing degrees of freedom. The gain in CG often comes at expense non-protein like local features. This could cause problems when transitioning full atom models a hierarchical framework. Here, energy function was validated applying it problem loop prediction. A novel method sample backbone atoms benchmarked using standard test set consisting 351 distinct loops....

A general understanding of the complex phenomenon protein evolution requires accurate description constraints that define sub-space proteins with mutations do not appreciably reduce fitness organism. Such can have multiple origins, in this work we present a model for constrained evolutionary trajectories represented by Markovian process throughout set protein-like structures artificially constructed to be topological intermediates between structure two natural occurring proteins. The number...

Abstract Automation and factorial experimental design together with cell-free in vitro transcription-translation systems offers a new route to the precise characterization of regulatory components. This now presents opportunity illuminate genetic circuitry from arcane microbial chassis, which are difficult assess vivo . One such host, Bacillus megaterium , is giant microbe industrial potential as producer recombinant proteins at gram per litre scale. Herein, we establish B. platform...

Synthetic networks require complex intertwined genetic regulation often relying on transcriptional activation or repression of target genes. CRISPRi-based transcription factors facilitate the programmable modulation endogenous synthetic promoter activity and process can be optimised by using software to select appropriate gRNAs limit non-specific gene modulation. Here, we develop a computational pipeline, gDesigner, that enables automated selection orthogonal with minimized off-target...