Design of a tunable bacterial gene expression system using engineered σ factors.

Extracytoplasmic function (ECF) σ factors selectively upregulate expression of specific genes in bacteria. These σ factors, belonging to the σ70 family, are much smaller than the primary, housekeeping σ factor with two helical domains that interact with the Pribnow box and the -35 element of the promoter DNA. Structural studies reveal that promoter specificity in a σ factor is determined by the interactions between a loop (L3) and the Pribnow box element. Similarly, the efficiency of transcription initiation is governed by the polypeptide linker between the two promoter-binding domains. Both these polypeptide segments are dynamic and poorly conserved among ECF σ factor homologs. This feature hitherto limited insights from protein-DNA interactions to be correlated with transcription initiation efficiency. Here, we describe an approach to characterize these features that govern the dynamic range of gene expression using chimeric Escherichia coli σE. The L3 loop and linker polypeptides in these σE chimeras were replaced by the corresponding segments from 10 annotated and functional Mycobacterium tuberculosis ECF σ's. In vitro and in vivo measurements to determine the effect of these polypeptide replacements provided an experimentally validated σE chimera- gene expression level data set. We illustrate the utility of this chimeric σE library in improving the efficiency of a biosynthetic pathway in E. coli. In a two-enzyme step, unaffected by feedback inhibition and substrate concentration, we show an increase in desired product levels by altering the relative intracellular levels of the target enzymes using this library of σ factors. The chimeric σE library thus demonstrates the feasibility of engineering σ factors to achieve bespoke expression levels of target genes for diverse applications in synthetic microbiology. IMPORTANCE: The synthesis of organic compounds involves the action of multiple enzymes in a biosynthetic pathway. Incorporating such biosynthetic pathways into microbes often leads to substantial cellular and metabolic stress resulting in low titers of the target compound. This limitation can be offset, in part, by optimizing enzyme efficiency and cellular enzyme concentration. The former involves significant efforts to achieve improvements in catalytic efficiency with the caveat that the metabolic load on a microbial cell imposed by the overexpression of the exogenous enzyme could result in reduced cell fitness. Here, we demonstrate the feasibility of engineered σ factors to modulate gene expression levels without significant genetic engineering. We note that changing the sequence of two flexible polypeptide loops without any changes to the structural scaffold of the transcription initiation factor σE could modulate the expression levels of the target genes. This ability provides a route to improve the efficiency of a biosynthetic pathway without altering the overall genomic makeup. The σE chimera library thus provides an avenue for pre-determined conditional gene expression of specific genes in Escherichia coli.

Dynamic Covalent Polyurethane Network Materials: Synthesis and Self-Healability.

Polyurethane (PU) has not only been widely used in the daily lives, but also extensively explored as an important class of the essential polymers for various applications. In recent years, significant efforts have been made on the development of self-healable PU materials that possess high performance, extended lifetime, great reliability, and recyclability. A promising approach is the incorporation of covalent dynamic bonds into the design of PU covalently crosslinked polymers and thermoplastic elastomers that can dissociate and reform indefinitely in response to external stimuli or autonomously. This review summarizes various strategies to synthesize self-healable, reprocessable, and recyclable PU materials integrated with dynamic (reversible) Diels-Alder cycloadduct, disulfide, diselenide, imine, boronic ester, and hindered urea bond. Furthermore, various approaches utilizing the combination of dynamic covalent chemistries with nanofiller surface chemistries are described for the fabrication of dynamic heterogeneous PU composites.

Macromolecularly Engineered Thermoreversible Heterogeneous Self-Healable Networks Encapsulating Reactive Multidentate Block Copolymer-Stabilized Carbon Nanotubes.

The development of heterogeneous covalent adaptable networks (CANs) embedded with carbon nanotubes (CNTs) that undergo reversible dissociation/recombination through thermoreversibility has been significantly explored. However, the carbon nanotube (CNT)-incorporation methods based on physical mixing and chemical modification could result in either phase separation due to structural incompatibility or degrading conjugation due to a disruption of π-network, thus lowering their intrinsic charge transport properties. To address this issue, the versatility of a macromolecular engineering approach through thermoreversibility by physical modification of CNT surfaces with reactive multidentate block copolymers (rMDBCs) is demonstrated. The formed CNTs stabilized with rMDBCs (termed rMDBC/CNT colloids) bearing reactive furfuryl groups is functioned as a multicrosslinker that reacts with a polymaleimide to fabricate robust heterogeneous polyurethane (PU) networks crosslinked through dynamic Diels-Alder (DA)/retro-DA chemistry. Promisingly, the fabricated PU network gels in which CNTs through rMDBC covalently embedded are flexible and robust to be bendable as well as exhibit self-healing elasticity and enhanced conductivity.

Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2.

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

Self-Healable Reprocessable Triboelectric Nanogenerators Fabricated with Vitrimeric Poly(hindered Urea) Networks.

In recent years, the advent of highly deformable and healable electronics is exciting and promising for next-generation electronic devices. In particular, self-healable triboelectric nanogenerators (SH-TENGs) serve as promising candidates based on the combination of the triboelectric effect, electrostatic induction, and self-healing action. However, the majority of SH-TENGs have been devised with weak polymeric networks that are healed with reversible supramolecular interactions or disulfides, thus resulting in poor mechanical properties and low resistance to creeping. To address this issue, we demonstrate the integration of mechanically strong and self-healable poly(hindered urea) (PHU) network in the fabrication of effective TENGs. The designed PHU network is flexible but shows greater mechanical property of tensile strength as high as 1.7 MPa at break. The network is capable of self-healing quickly and repeatedly as well as being reprocessable under mild conditions, enabling the recovery of triboelectric performances after the complete healing of the damaged surfaces. Furthermore, the interfacial-polarization-induced enhancement of dielectric constant endows our SH-TENG with the highest triboelectric output performance (169.9 V/cm2) among the reported healable TENGs. This work presents an avenue to the development of mechanical energy-harvesting devices and self-powered sensors with excellent stretchability, high recoverability, and good mechanical strength.

Microfluidic Assembly To Synthesize Dual Enzyme/Oxidation-Responsive Polyester-Based Nanoparticulates with Controlled Sizes for Drug Delivery.

Controlling the size and narrow size distribution of polymer-based nanocarriers for targeted drug delivery is an important parameter that significantly influences their colloidal stability, biodistribution, and targeting ability. Herein, we report a high-throughput microfluidic process to fabricate colloidally stable aqueous nanoparticulate colloids with tunable sizes at 50-150 nm and narrow size distribution. The nanoparticulates are designed with different molecular weight polyesters having both ester bonds (responsive to esterase) and sulfide linkages (to oxidative reaction) on the backbones, thus exhibiting dual esterase/oxidation responses, causing the destabilization of the nanoparticulates to lead to the controlled release of encapsulated therapeutics. The systematic investigation on both microfluidic and formulation parameters enables to control their properties as allowing for decreasing nanoparticulate sizes as well as improving colloidal stability and cytotoxicity. Further to such control over smaller size and narrow size distribution, dual stimuli-responsive degradation and excellent cellular uptake could suggest that the microfluidic nanoparticulates stabilized with polymeric stabilizers could offer the versatility toward dual smart drug delivery exhibiting enhanced release kinetics.

Thermally Labile Self-Healable Branched Gel Networks Fabricated by New Macromolecular Engineering Approach Utilizing Thermoreversibility.

A new approach based on macromolecular engineering through thermoreversibility is reported to fabricate the engineered gel networks of thermally labile branched polymers exhibiting robust self-healing. This approach centers on the synthesis of linear polymers having Diels-Alder cycloadducts in the backbones (DALPs) through A2 + B2 step-growth polymerization of a difunctional furan and a difunctional maleimide. Reactive mixtures of the resulting DALP with a polyfuran at elevated temperature allow for the formation of engineered gel networks through random dissociation of backbone DA linkages of the DALPs by retro-Diels-Alder reaction, followed by their reconstruction in the presence of polyfuran (with functionality > 2) by Diels-Alder reaction. Optimizing the ratio of furan to DA linkages in the reactive mixtures yields thermally labile networks exhibiting excellent thermoreversibility. Effective self-healing demonstrated with reconstruction from two separate pieces and complete void filling on surface cuts as well as recovery of healing viscoelasticity suggest that the new macromolecular engineering approach offers versatility toward the development of thermally mendable thermosets.