Electro‐Bio‐Fabrication: Diverse Routes To Functional Films/Hydrogels

Please click Additional Files below to see the full abstract

Repurposing E. coli by Engineering Quorum Sensing and Redox Genetic Circuits

Because cells have the extraordinary ability to sense and respond to even subtle environmental changes by intricately regulating their gene expression patterns, their behaviors can be intentionally “tuned” by altering the state of their environments in a prescribed or rational manner. Rational control of both external and internal molecular stimuli provides a basis for many biotechnological applications including the expression of foreign protein products. This is done by coordinately controlling product synthesis while retaining the cell in a productive state. Quorum sensing (QS), a molecular signaling modality that mediates cell-cell communication, autonomously facilitates both inter- and intra-species gene regulation. This process can be rewired to enable autonomously actuated, but molecularly programmed, genetic control. Recently, even electrical signals, which have long been used to control the most sophisticated of man-made devices, are now employed to alter cell signaling processes enabling computer programmed behavior, particularly in cells suitably engineered to accommodate electrical signals. By minimally engineering these genetic circuits, new applications have emerged for the repurposing of Escherichia coli, from creating innovative sensor concepts to stimulating the emerging field of electrogenetics

Electrogenetic actuation of gene expression in bacteria: Towards programmable biological function based on molecular signaling

The ability to interconvert information between electronic and biologic systems has already transformed our ability to record and actuate biological function (e.g., EEG, EKG, defibrillators). In parallel, we have begun to demand biological connectivity from electronic consumer products (fitbitTM, cell phones, etc.). There are significant gaps, however, that must be overcome before biological information can be seamlessly conveyed and before biological function can be electronically “programmed”. A communication gap exists whereby the common vectors for information flow in biology are ions and molecules; they are electrons and photons in electronics. Since there are essentially no “free” electrons in biological systems, there is essentially no direct “translator” of electrons to molecules and vice versa. Gaining access to molecular communication is essential as molecules are the primary vector that drives biological function. There is also a fabrication gap to overcome. It is difficult to construct microelectronic devices that include labile biological components. We are developing tools of “biofabrication” that enable facile assembly of biological components within devices that preserve their native biological function. By recognizing that biological redox active molecules are a biological equivalent of an electron-carrying wire, we have developed biological surrogates for electronic devices, including a biological redox capacitor. We have also turned to synthetic biology to provide a means to sample, interpret and report on biological information contained in molecular communications circuitry. Finally, we have developed synthetic genetic circuits that enable electronic actuation of gene expression. This presentation will introduce the concepts of molecular communication that are enabled by integrating relatively simple concepts in synthetic biology with biofabrication. Our presentation will show how engineered cells represent a versatile means for mediating the molecular “signatures” commonly found in complex environments, or in other words, they are conveyors of molecular communication

Transition From Unilamellar to Bilameller Vesicles Induced by an Amphiphilic Biopolymer

We report some unusual structural transitions upon the addition of an amphiphilic biopolymer to unilamellar surfactant vesicles. The polymer is a hydrophobically modified chitosan and it embeds its hydrophobes in vesicle bilayers. We study vesicle-polymer mixtures using small-angle neutron scattering (SANS) and cryotransmission electron microscopy (cryo-TEM). When low amounts of the polymer are added to unilamellar vesicles of ca. 120 nm diameter, the vesicle size decreases by about 50%. Upon further addition of polymer, lamellar peaks are observed in the SANS spectra at high scattering vectors. We show that these spectra correspond to a co-existence of unilamellar and bilamellar vesicles. The transition to bilamellar vesicles as well as the changes in unilamellar vesicle size are further confirmed by cryo-TEM. A mechanism for the polymer-induced transitions in vesicle morphology is proposed

Psychometric Evaluation of a Coping Strategies Inventory Short-Form (CSI-SF) in the Jackson Heart Study Cohort

This study sought to establish the psychometric properties of a Coping Strategies Inventory Short Form (CSISF) by examining coping skills in the Jackson Heart Study cohort. We used exploratory and confirmatory factor analysis, Pearson’s correlation, and Cronbach Alpha to examine reliability and validity in the CSI-SF that solicited responses from 5302 African American men and women between the ages of 35 and 84. One item was dropped from the 16-item CSI-SF, making it a 15-item survey. No significant effects were found for age and gender, strengthening the generalizability of the CSI-SF. The internal consistency reliability analysis revealed reliability between alpha = 0.58-0.72 for all of the scales, and all of the fit indices used to examine the CSI-SF provided support for its use as an adequate measure of coping. This study provides empirical support for utilizing this instrument in future efforts to understand the role of coping in moderating health outcomes

Correction: Addison, C.C., et al. Psychometric Evaluation of a Coping Strategies Inventory Short-Form (CSI-SF) in the Jackson Heart Study Cohort. Int. J. Environ. Res. Public Health 2007, 4, 243–249.

We found some errors in Table 4 in our paper published in the International Journal of Environmental Research and Public Health recently [1]. Table 4 is corrected as follows: [...

Genes and lipids that impact uptake and assimilation of exogenous coenzyme Q in Saccharomyces cerevisiae.

Coenzyme Q (CoQ) is an essential player in the respiratory electron transport chain and is the only lipid-soluble antioxidant synthesized endogenously in mammalian and yeast cells. In humans, genetic mutations, pathologies, certain medical treatments, and aging, result in CoQ deficiencies, which are linked to mitochondrial, cardiovascular, and neurodegenerative diseases. The only strategy available for these patients is CoQ supplementation. CoQ supplements benefit a small subset of patients, but the poor solubility of CoQ greatly limits treatment efficacy. Consequently, the efficient delivery of CoQ to the mitochondria and restoration of respiratory function remains a major challenge. A better understanding of CoQ uptake and mitochondrial delivery is crucial to make this molecule a more efficient and effective therapeutic tool. In this study, we investigated the mechanism of CoQ uptake and distribution using the yeast Saccharomyces cerevisiae as a model organism. The addition of exogenous CoQ was tested for the ability to restore growth on non-fermentable medium in several strains that lack CoQ synthesis (coq mutants). Surprisingly, we discovered that the presence of CoQ biosynthetic intermediates impairs assimilation of CoQ into a functional respiratory chain in yeast cells. Moreover, a screen of 40 gene deletions considered to be candidates to prevent exogenous CoQ from rescuing growth of the CoQ-less coq2Δ mutant, identified six novel genes (CDC10, RTS1, RVS161, RVS167, VPS1, and NAT3) as necessary for efficient trafficking of CoQ to mitochondria. The proteins encoded by these genes represent essential steps in the pathways responsible for transport of exogenously supplied CoQ to its functional sites in the cell, and definitively associate CoQ distribution with endocytosis and intracellular vesicular trafficking pathways conserved from yeast to human cells

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.https://doi.org/10.1186/s12934-021-01703-

The XMM-Newton survey of the Small Magellanic Cloud: XMMUJ005011.2-730026 = SXP214, a Be/X-ray binary pulsar

In the course of the XMM-Newton survey of the Small Magellanic Cloud (SMC), a region to the east of the emission nebula N19 was observed in November 2009. To search for new candidates for high mass X-ray binaries the EPIC PN and MOS data of the detected point sources were investigated and their spectral and temporal characteristics identified. A new transient (XMMUJ005011.2-730026= SXP214) with a pulse period of 214.05 s was discovered; the source had a hard X-ray spectrum with power-law index of ~0.65. The accurate X-ray source location permits the identification of the X-ray source with a ~15th magnitude Be star, thereby confirming this system as a new Be/X-ray binary.Comment: 8 pages 11 figures. Accepted for publication in MNRA

A Redox-Based Autoinduction Strategy to Facilitate Expression of 5xCys-Tagged Proteins for Electrobiofabrication

Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices