synthesis-infectious-bacteriophages-an-e-coli-based-cell-free

Synthesis of Infectious Bacteriophages in an <em>E. coli-</em>based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

University of Minnesota

The optimal management strategy for acute Achilles tendon rupture is controversial.

Operative versus nonoperative management of acute Achilles tendon rupture: expected-value decision analysis.

Lipid A is the causative agent of Gram-negative sepsis, a leading cause of mortality among hospitalized patients. Compounds that bind lipid A can limit its detrimental effects. Polymyxin B, a cationic peptide antibiotic, is one of the simplest molecules capable of selectively binding lipid A and may serve as a model for further development of lipid A binding agents. However, association of polymyxin B with lipid A is not fully understood, primarily due to the low solubility of lipid A in water and inhomogeneity of lipid A preparations. To better understand lipid A-polymyxin B interaction, pure lipid A derivatives were prepared with incrementally varied lipid chain lengths. These compounds proved to be more soluble in water than lipid A, with higher aggregation concentrations. Isothermal titration calorimetric studies of these lipid A derivatives with polymyxin B and polymyxin B nonapeptide indicate that binding stoichiometries (peptide to lipid A derivative) are less than 1 and that affinities of these binding partners correlate with the aggregation states of the lipid A derivatives. These studies also suggest that cooperative ionic interactions dominate association of polymyxin B and polymyxin B nonapeptide with lipid A.

Synthesis of lipid A derivatives and their interactions with polymyxin B and polymyxin B nonapeptide.

Crohn's disease and ulcerative colitis are the primary inflammatory bowel diseases (IBDs) affecting the gastrointestinal tract. The current therapy aims at decreasing inflammation and reducing symptoms. This typically requires immune suppression by steroids, thiopurines, methotrexate, or tumor necrosis factor inhibitors. Patients may be unreceptive to medical therapy, and some may discontinue the treatment due to adverse effects. Noninvasive, transcutaneous vagus nerve stimulation (VNS) is currently used as a treatment for depression and epilepsy, and it is being investigated for the treatment of conditions such as multiple sclerosis, migraines, and Alzheimer's disease. Recent studies have demonstrated the importance of splenic and vagus nerve functions in the inflammatory process through the production of certain cytokines. We hypothesize that using transcutaneous VNS via the auricular afferent branch could achieve a selective anti-inflammatory effect on the intestinal wall. This review examines the possibility of using vagal stimulators as a therapy for IBD. This could open the door to novel treatments for numerous vagally mediated diseases characterized by poor responses to current therapies.

Bioelectrical Stimulation for the Reduction of Inflammation in Inflammatory Bowel Disease.

We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.

The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.

Understanding the role of basal bodies (BBs) during development and disease has been largely overshadowed by research into the function of the cilium. Although these two organelles are closely associated, they have specific roles to complete for successful cellular development. Appropriate development and function of the BB are fundamental for cilia function. Indeed, there are a growing number of human genetic diseases affecting ciliary development, known collectively as the ciliopathies. Accumulating evidence suggests that BBs establish cell polarity, direct ciliogenesis, and provide docking sites for proteins required within the ciliary axoneme. Major contributions to our knowledge of BB structure and function have been provided by studies in flagellated or ciliated unicellular eukaryotic organisms, specifically Tetrahymena and Chlamydomonas. Reproducing these and other findings in vertebrates has required animal in vivo models. Zebrafish have fast become one of the primary organisms of choice for modeling vertebrate functional genetics. Rapid ex-utero development, proficient egg laying, ease of genetic manipulation, and affordability make zebrafish an attractive vertebrate research tool. Furthermore, zebrafish share over 80 % of disease causing genes with humans. In this article, we discuss the merits of using zebrafish to study BB functional genetics, review current knowledge of zebrafish BB ultrastructure and mechanisms of function, and consider the outlook for future zebrafish-based BB studies.

Zebrafish: a vertebrate tool for studying basal body biogenesis, structure, and function.

Surgeons need to understand the expected outcomes for super obese patients undergoing tracheotomy to appropriately counsel patients and families about likely risks and benefits.

Tracheotomy Outcomes in Super Obese Patients.

Giant cutaneous squamous cell carcinoma requiring emergent embolization.

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids.

Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137-2141. © 2017 Wiley Periodicals, Inc.

Ryan Marshall

School of Physics and Astronomy

University of Minnesota

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Ryan Marshall

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Physics and Astronomy

University of Minnesota

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

School of Physics and Astronomy