Elucidation of Sequence-Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli.

Transcription factor (TF)-promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF-promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF-promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library's distribution characteristics, we elucidate sequence-function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence-function relationship of a σ54-dependent, butanol-responsive TF-promoter pair, BmoR-PBMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized PBMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in PBMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ54-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.

Transcription factor-based biosensors and inducible systems in non-model bacteria: current progress and future directions.

Genetic diversity within the geobiosphere encompasses enormous sensing capabilities and many non-model bacteria are of biotechnological interest. Biosensing, or more generally inducible, systems are a vital component of metabolic engineering, as they allow tight control of gene expression as well as the basis for high-throughput screens on non-growth-related phenotypes. While these inducible systems, primarily transcription factor/promoter pairs, have been utilized extensively in Escherichia coli, progress in other bacteria is limited because of differences in transcription machinery, physiological compatibility of parts and proteins, and other nuances. Here, we provide an overview of the available genetic biosensing elements in non-model organisms and state-of-the-art efforts to engineer them, and then discuss challenges preventing these methods from common use in non-model bacteria.

Recent Developments of the Synthetic Biology Toolkit for Clostridium.

The Clostridium genus is a large, diverse group consisting of Gram-positive, spore-forming, obligate anaerobic firmicutes. Among this group are historically notorious pathogens as well as several industrially relevant species with the ability to produce chemical commodities, particularly biofuels, from renewable biomass. Additionally, other species are studied for their potential use as therapeutics. Although metabolic engineering and synthetic biology have been instrumental in improving product tolerance, titer, yields, and feed stock consumption capabilities in several organisms, low transformation efficiencies and lack of synthetic biology tools and genetic parts make metabolic engineering within the Clostridium genus difficult. Progress has recently been made to overcome challenges associated with engineering various Clostridium spp. For example, developments in CRISPR tools in multiple species and strains allow greater capability to produce edits with greater precision, faster, and with higher efficiencies. In this mini-review, we will highlight these recent advances and compare them to established methods for genetic engineering in Clostridium. In addition, we discuss the current state and development of Clostridium-based promoters (constitutive and inducible) and reporters. Future progress in this area will enable more rapid development of strain engineering, which would allow for the industrial exploitation of Clostridium for several applications including bioproduction of several commodity products.

Selective TNF inhibition for chronic stroke and traumatic brain injury: an observational study involving 629 consecutive patients treated with perispinal etanercept.

BACKGROUND: Brain injury from stroke and traumatic brain injury (TBI) may result in a persistent neuroinflammatory response in the injury penumbra. This response may include microglial activation and excess levels of tumour necrosis factor (TNF). Previous experimental data suggest that etanercept, a selective TNF inhibitor, has the ability to ameliorate microglial activation and modulate the adverse synaptic effects of excess TNF. Perispinal administration may enhance etanercept delivery across the blood-CSF barrier. OBJECTIVE: The objective of this study was to systematically examine the clinical response following perispinal administration of etanercept in a cohort of patients with chronic neurological dysfunction after stroke and TBI. METHODS: After approval by an independent external institutional review board (IRB), a chart review of all patients with chronic neurological dysfunction following stroke or TBI who were treated open-label with perispinal etanercept (PSE) from November 1, 2010 to July 14, 2012 at a group medical practice was performed. RESULTS: The treated cohort included 629 consecutive patients. Charts of 617 patients following stroke and 12 patients following TBI were reviewed. The mean age of the stroke patients was 65.8 years ± 13.15 (range 13-97). The mean interval between treatment with PSE and stroke was 42.0 ± 57.84 months (range 0.5-419); for TBI the mean interval was 115.2 ± 160.22 months (range 4-537). Statistically significant improvements in motor impairment, spasticity, sensory impairment, cognition, psychological/behavioural function, aphasia and pain were noted in the stroke group, with a wide variety of additional clinical improvements noted in individuals, such as reductions in pseudobulbar affect and urinary incontinence. Improvements in multiple domains were typical. Significant improvement was noted irrespective of the length of time before treatment was initiated; there was evidence of a strong treatment effect even in the subgroup of patients treated more than 10 years after stroke and TBI. In the TBI cohort, motor impairment and spasticity were statistically significantly reduced. DISCUSSION: Irrespective of the methodological limitations, the present results provide clinical evidence that stroke and TBI may lead to a persistent and ongoing neuroinflammatory response in the brain that is amenable to therapeutic intervention by selective inhibition of TNF, even years after the acute injury. CONCLUSION: Excess TNF contributes to chronic neurological, neuropsychiatric and clinical impairment after stroke and TBI. Perispinal administration of etanercept produces clinical improvement in patients with chronic neurological dysfunction following stroke and TBI. The therapeutic window extends beyond a decade after stroke and TBI. Randomized clinical trials will be necessary to further quantify and characterize the clinical response.