Improving Biomanufacturing Production with Novel ELP-Based Transcriptional Regulators

Microbes can be used to produce valuable drugs, chemicals, and biofuels, but their potential has not been fully realized due to low production yields. To improve biomanufacturing processes and yield, we are developing novel, transcriptional regulators using biosynthesis technology in order to improve cellular health and overall production. Our regulator contains elastin-like polypeptides (ELPs), which make ideal sensors since they exhibit a sharp, inverse phase transition to indicators of cell health such as intracellular pH and ionic strength, and external stimuli such as temperature. We hypothesize that ELP can be fused to transcription factors to control expression of target genes. As proof of concept, Tet repressor protein (TetR) was fused to ELP to control expression of the red fluorescent protein mCherry, which was cloned under a Tet repressible promoter. The ability of TetR-ELP to control expression was determined by measuring fluorescence above and below the transition temperature (Tt) of ELP. Below the Tt, TetR is free to repress mCherry production, but above the Tt, ELP should aggregate, preventing TetR from repressing mCherry. However, our results showed that fluorescence was not affected as expected. We hypothesize that the observed behavior is due to either TetR having temperature sensitivity or that the binding affinity of TetR to DNA is much stronger than ELP aggregation. Further steps include proving this hypothesis and finding alternative transcription factors to test. These tested gene regulators will allow us to optimize production yield of microbe and bring development in manufacturing of drugs, chemicals, and biofuels

Using Elastin-Like Polypeptides for Better Retention of Biofuels

Elastin-like polypeptides (ELPs) are synthetic molecules that exhibit an interesting property of inverse temperature phase transition; they exist as soluble monomers at low temperatures and form insoluble aggregates at higher temperatures. The transition temperature depends on the pH, salt concentration, and the amino acid sequence of the ELP. This unique and reversible behavior, along with their high biocompatibility has made them a strategic tool for various biomedical applications. However, their hydrophobic properties also make them a prime candidate for biofuel production. As high levels of many commercially important organic solvents are toxic to the cells that make them, ELPs can potentially alleviate the strain on cells by aggregating around the hydrophobic product. ELP’s are simply purified by exploiting their phase transition property and through serial centrifugation at different temperatures. The retention of various bio-products and the cell survivability was analyzed for E. coli containing ELPs both in vivo and in vitro. Confocal microscopy and fluorescence measurements were used to verify the results. The present study provides proof of principle that ELPs have high affinity with certain commercially important biologics and can be a strategic tool to increase their yield

Improving Biomanufacturing Production with Tunable Transcriptional Regulation via Elastin-like Polypeptides

The metabolism of E. coli and other microbes can be engineered to create valuable chemicals such as biofuels, medicines, etc. However, process efficiency is limited by the toxicity of intermediates in the production pathway, which induces cellular stress and killing the production in cells. By fusing elastin-like polypeptides (ELPs) with sigma factors (SF), we propose a stress feedback system can be created to recognize cues of cellular health and autoregulate expression of bioproduction pathways for improved health and production. ELPs undergo a sharp, reversible, phase transition causing an aggregation above a certain temperature (Tt) based on conditions that align with intracellular health such as intracellular pH. This behavior, along with the ability to control Tt through sequence alterations, makes ELPs ideal sensors for controlling gene expression. Fused SF, which activated gene expression, are sequestered in ELP-SF aggregates above the transition temperature, reducing their free concentration. To evaluate the potential of ELP-SF to control gene expression, we expressed green fluorescent protein (GFP) from a promoter driven by the fused SF. In vivo, this system activated the expression of GFP at levels comparable to a SF control. However, at elevated temperatures, the system reduces gene expression by 20% relative to the control demonstrating the ability of the construct to control gene expression. The dynamic performance of the system was also modeled in MATLAB to reveal key parameters that affect system behavior. These results validate our main hypothesis and suggest a new strategy to optimize the sustainable production of valuable chemicals from microbes

Cost-effective Paper-based Diagnostic Using Split Proteins to Detect Yeast Infections

The common yeast infection, vulvovaginal candidiasis, affects three out of four women throughout their lifetime and can be spread to their child in the form of oral candidiasis (thrush). This disease is caused by the fungal pathogen Candida albicans, which is also a major cause of systemic candidiasis, a rarer but deadly disease with up to a 49% lethality rate. Current widely-used diagnostic methods include cell cultures, pH tests, and antibody detection, to assist effective treatment. Despite availability of various diagnostic methods, there is no inexpensive, rapid, and accurate way to detect C. albicans infection. This project aims to develop a paper-based diagnostic test for C. albicans that is, cost-effective, quick, and precise. The test detects the specific biomarkers farnesol and tyrosol produced by C. albicans by binding them to the split proteins pqsR and tyrosinase, respectively. Upon binding, a split horseradish peroxidase catalyzes and produces an amplified colorimetric signal by oxidizing the substrate tetramethylbenzidine (TMB) turning the paper blue. This test will produce a colorimetric output for a simple-to-understand diagnosis without any infrastructure. We predict that this device can give a response in under 2 minutes while costing around an estimated 10 cents per device This test may provide a way for an easy and cheap way to diagnose candidiasis worldwide, reducing the abuse of antifungals and provide an accurate way to treat vulvovaginal candidiasis and systemic candidiasis

Cost-effective Paper-based Diagnostic Using Split Proteins to Detect Yeast Infections

The common yeast infection, vulvovaginal candidiasis, affects three out of four women throughout their lifetime and can be spread to their child in the form of oral candidiasis (thrush). This disease is caused by the fungal pathogen Candida albicans, which is also a major cause of systemic candidiasis, a rarer but deadly disease with up to a 49% lethality rate. Current widely-used diagnostic methods include cell cultures, pH tests, and antibody detection, to assist effective treatment. Despite availability of various diagnostic methods, there is no inexpensive, rapid, and accurate way to detect C. albicans infection. This project aims to develop a paper-based diagnostic test for C. albicans that is, cost-effective, quick, and precise. The test detects the specific biomarkers farnesol and tyrosol produced by C. albicans by binding them to the split proteins pqsR and tyrosinase, respectively. Upon binding, a split horseradish peroxidase catalyzes and produces an amplified colorimetric signal by oxidizing the substrate tetramethylbenzidine (TMB) turning the paper blue. This test will produce a colorimetric output for a simple-to-understand diagnosis without any infrastructure. We predict that this device can give a response in under 2 minutes while costing around an estimated 10 cents per device This test may provide a way for an easy and cheap way to diagnose candidiasis worldwide, reducing the abuse of antifungals and provide an accurate way to treat vulvovaginal candidiasis and systemic candidiasis

Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes

available in PMC 2016 November 07The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing.United States. Dept. of Energy. Office of Science (Biological and Environmental Research (BER) program)United States. Department of Energy (DOE Grant DE-SC0010352)United States. Department of Agriculture (Award 2011-67017-20459)Institute for Collaborative Biotechnologies (grant W911NF-09-0001

Outcomes in patients with gunshot wounds to the brain.

Introduction:Gunshot wounds to the brain (GSWB) confer high lethality and uncertain recovery. It is unclear which patients benefit from aggressive resuscitation, and furthermore whether patients with GSWB undergoing cardiopulmonary resuscitation (CPR) have potential for survival or organ donation. Therefore, we sought to determine the rates of survival and organ donation, as well as identify factors associated with both outcomes in patients with GSWB undergoing CPR. Methods:We performed a retrospective, multicenter study at 25 US trauma centers including dates between June 1, 2011 and December 31, 2017. Patients were included if they suffered isolated GSWB and required CPR at a referring hospital, in the field, or in the trauma resuscitation room. Patients were excluded for significant torso or extremity injuries, or if pregnant. Binomial regression models were used to determine predictors of survival/organ donation. Results:825 patients met study criteria; the majority were male (87.6%) with a mean age of 36.5 years. Most (67%) underwent CPR in the field and 2.1% (n=17) survived to discharge. Of the non-survivors, 17.5% (n=141) were considered eligible donors, with a donation rate of 58.9% (n=83) in this group. Regression models found several predictors of survival. Hormone replacement was predictive of both survival and organ donation. Conclusion:We found that GSWB requiring CPR during trauma resuscitation was associated with a 2.1% survival rate and overall organ donation rate of 10.3%. Several factors appear to be favorably associated with survival, although predictions are uncertain due to the low number of survivors in this patient population. Hormone replacement was predictive of both survival and organ donation. These results are a starting point for determining appropriate treatment algorithms for this devastating clinical condition. Level of evidence:Level II

Synthetic Metabolism: Engineering Biology at the Protein and Pathway Scales

Biocatalysis has become a powerful tool for the synthesis of high-value compounds, particularly so in the case of highly functionalized and/or stereoactive products. Nature has supplied thousands of enzymes and assembled them into numerous metabolic pathways. Although these native pathways can be use to produce natural bioproducts, there are many valuable and useful compounds that have no known natural biochemical route. Consequently, there is a need for both unnatural metabolic pathways and novel enzymatic activities upon which these pathways can be built. Here, we review the theoretical and experimental strategies for engineering synthetic metabolic pathways at the protein and pathway scales, and highlight the challenges that this subfield of synthetic biology currently faces.Synthetic Biology Engineering Research CenterNational Science Foundation (Grant no. 0540879

Diabetes and risk of pancreatic cancer: a pooled analysis from the pancreatic cancer cohort consortium

Diabetes is a suspected risk factor for pancreatic cancer, but questions remain about whether it is a risk factor or a result of the disease. This study prospectively examined the association between diabetes and the risk of pancreatic adenocarcinoma in pooled data from the NCI pancreatic cancer cohort consortium (PanScan). The pooled data included 1,621 pancreatic adenocarcinoma cases and 1,719 matched controls from twelve cohorts using a nested case-control study design. Subjects who were diagnosed with diabetes near the time (< 2 years) of pancreatic cancer diagnosis were excluded from all analyses. All analyses were adjusted for age, race, gender, study, alcohol use, smoking, BMI, and family history of pancreatic cancer. Self-reported diabetes was associated with a forty percent increased risk of pancreatic cancer (OR = 1.40, 95 % CI: 1.07, 1.84). The association differed by duration of diabetes; risk was highest for those with a duration of 2-8 years (OR = 1.79, 95 % CI: 1.25, 2.55); there was no association for those with 9+ years of diabetes (OR = 1.02, 95 % CI: 0.68, 1.52). These findings provide support for a relationship between diabetes and pancreatic cancer risk. The absence of association in those with the longest duration of diabetes may reflect hypoinsulinemia and warrants further investigation