Augmenting Protocols for In-situ Separation of Biocompounds.

136388-Thumbnail Image.png
Description
In our modern world the source of for many chemicals is to acquire and refine oil. This process is becoming an expensive to the environment and to human health. Alternative processes for acquiring the final product have been developed but…

In our modern world the source of for many chemicals is to acquire and refine oil. This process is becoming an expensive to the environment and to human health. Alternative processes for acquiring the final product have been developed but still need work. One product that is valuable is butanol. The normal process for butanol production is very intensive but there is a method to produce butanol from bacteria. This process is better because it is more environmentally safe than using oil. One problem however is that when the bacteria produce too much butanol it reaches the toxicity limit and stops the production of butanol. In order to keep butanol from reaching the toxicity limit an adsorbent is used to remove the butanol without harming the bacteria. The adsorbent is a mesoporous carbon powder that allows the butanol to be adsorbed on it. This thesis explores different designs for a magnetic separation process to extract the carbon powder from the culture.
Date Created
2015-05
Agent

Investigation of the effect of efflux pumps on the toxicity of phenol, 2-phenylethanol, and styrene to E. coli

136282-Thumbnail Image.png
Description
Depletion of fossil fuel resources has led to the investigation of alternate feedstocks for and methods of chemical synthesis, in particular the use of E. coli biocatalysts to produce fine commodity chemicals from renewable glucose sources. Production of phenol, 2-phenylethanol,…

Depletion of fossil fuel resources has led to the investigation of alternate feedstocks for and methods of chemical synthesis, in particular the use of E. coli biocatalysts to produce fine commodity chemicals from renewable glucose sources. Production of phenol, 2-phenylethanol, and styrene was investigated, in particular the limitation in yield and accumulation that results from high product toxicity. This paper examines two methods of product toxicity mitigation: the use of efflux pumps and the separation of pathways which produce less toxic intermediates. A library of 43 efflux pumps from various organisms were screened for their potential to confer resistance to phenol, 2-phenylethanol, and styrene on an E. coli host. A pump sourced from P. putida was found to allow for increased host growth in the presence of styrene as compared to a cell with no efflux pump. The separation of styrene producing pathway was also investigated. Cells capable of performing the first and latter halves of the synthesis were allowed to grow separately and later combined in order to capitalize on the relatively lower toxicity of the intermediate, trans-cinnamate. The styrene production and yield from this separated set of cultures was compared to that resulting from the growth of cells containing the full set of styrene synthesis genes. Results from this experiment were inconclusive.
Date Created
2015-05
Agent

Design and Engineering of Synthetic Gene Networks

155857-Thumbnail Image.png
Description
Synthetic gene networks have evolved from simple proof-of-concept circuits to

complex therapy-oriented networks over the past fifteen years. This advancement has

greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a

mechanism that cells use to achieve a discrete number…

Synthetic gene networks have evolved from simple proof-of-concept circuits to

complex therapy-oriented networks over the past fifteen years. This advancement has

greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a

mechanism that cells use to achieve a discrete number of mutually exclusive states in

response to environmental inputs. However, complex contextual connections of gene

regulatory networks in natural settings often impede the experimental establishment of

the function and dynamics of each specific gene network.

In this work, diverse synthetic gene networks are rationally designed and

constructed using well-characterized biological components to approach the cell fate

determination and state transition dynamics in multistable systems. Results show that

unimodality and bimodality and trimodality can be achieved through manipulation of the

signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to

communicate with each other.

Moreover, a synthetic quadrastable circuit is also built and experimentally

demonstrated to have four stable steady states. Experiments, guided by mathematical

modeling predictions, reveal that sequential inductions generate distinct cell fates by

changing the landscape in sequence and hence navigating cells to different final states.

Circuit function depends on the specific protein expression levels in the circuit.

We then establish a protein expression predictor taking into account adjacent

transcriptional regions’ features through construction of ~120 synthetic gene circuits

(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.

These combined results illustrate applications of synthetic gene networks to

understand the cell fate determination and state transition dynamics in multistable

systems. A protein-expression predictor is also developed to evaluate and tune circuit

dynamics.
Date Created
2017
Agent

Synthetic Biology Applications in Industrial Microbiology

128178-Thumbnail Image.png
Description

With the ability to perform a multitude of unique and complex chemical transformations, microorganisms have long been the “workhorses” of many industrial processes. However, in addition to exploiting the utility of naturally evolved phenotypes, the principles, strategies, and tools of…

With the ability to perform a multitude of unique and complex chemical transformations, microorganisms have long been the “workhorses” of many industrial processes. However, in addition to exploiting the utility of naturally evolved phenotypes, the principles, strategies, and tools of synthetic biology are now being applied to facilitate the engineering of tailor-made microbes capable of tackling some of society's most important and toughest challenges. Fueled in part by exponentially increasing reservoirs of bioinformatic data and coupled with more robust and powerful tools for its processing, research in the past decade has brought about new and broadened perspectives of fundamental biological phenomena. The application of said insight is now beginning to unlock the unprecedented potential of synthetic biology in biotechnology, as well as its considerable promise for addressing previously unsolved global challenges. For example, within the realm of industrial microbiology, progress in the field of synthetic biology has enabled the development of new biosynthetic pathways for the production of renewable fuels and chemicals, programmable logic controls to regulate and optimize complex cellular functions, and robust microbes for the destruction of harmful environmental contaminants. In this Research Topic, a collection of articles—including original research, reviews, and mini-reviews—from leading investigators in the synthetic biology community are presented to capture the current state, recent progress, and over-arching challenges associated with integrating synthetic biology with industrial microbiology and biotechnology.

Date Created
2014-08-26
Agent

Activity of Lactobacillus Brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors

128735-Thumbnail Image.png
Description

The R-specific alcohol dehydrogenase (ADH) from Lactobacillus brevis LB19 (LbADH) was studied with respect to its ability to reduce a series of 3- through 5-carbon 2-alkanones and aldehydes of relevance as biofuel precursors. Although active on all substrates tested, LbADH…

The R-specific alcohol dehydrogenase (ADH) from Lactobacillus brevis LB19 (LbADH) was studied with respect to its ability to reduce a series of 3- through 5-carbon 2-alkanones and aldehydes of relevance as biofuel precursors. Although active on all substrates tested, LbADH displays a marked preference for longer chain substrates. Interestingly, however, 2-alkanones were found to impose substrate inhibition towards LbADH, whereas aldehyde substrates rendered no such effect. Inhibition caused by 2-alkanones was furthermore found to intensify with increasing chain length. Despite demonstrating both primary and secondary ADH activities, a preliminary sequence analysis suggests that LbADH remains distinct from other, previously characterized primary-secondary ADHs. In addition to further characterizing the substrate range of this industrially important enzyme, this study suggests that LbADH has the potential to serve as a useful enzyme for the engineering of various novel alcohol biofuel pathways.

Date Created
2015-08-05
Agent

Modulation of mammalian cell behavior for enhancing polymer-mediated transgene expression

154922-Thumbnail Image.png
Description
Gene delivery is a broadly applicable tool that has applications in gene therapy, production of therapeutic proteins, and as a study tool to understand biological pathways. However, for successful gene delivery, the gene and its carrier must bypass or traverse…

Gene delivery is a broadly applicable tool that has applications in gene therapy, production of therapeutic proteins, and as a study tool to understand biological pathways. However, for successful gene delivery, the gene and its carrier must bypass or traverse a number of formidable obstacles before successfully entering the cell’s nucleus where the host cell’s machinery can be utilized to express a protein encoded by the gene of interest. The vast majority of work in the gene delivery field focuses on overcoming these barriers by creative synthesis of nanoparticle delivery vehicles or conjugation of targeting moieties to the nucleic acid or delivery vehicle, but little work focuses on modifying the target cell’s behavior to make it more amenable to transfection.

In this work, a number of kinase enzymes have been identified by inhibition to be targets for enhancing polymer-mediated transgene expression (chapter 2), including the lead target which appears to affect intracellular trafficking of delivered nucleic acid cargo. The subsequent sections (chapters 3 and 4) of this work focus on targeting epigenetic modifying enzymes to enhance polymer-mediated transgene expression, and a number of candidate enzymes have been identified. Some mechanistic evaluation of these targets have been carried out and discussion of ongoing experiments and future directions to better understand the mechanistic descriptions behind the phenomena are discussed. The overall goal is to enhance non-viral (polymer-mediated) transgene expression by modulating cellular behavior for general gene delivery applications.
Date Created
2016
Agent

Exploring biosynthetic pathways for aromatic ester production

154882-Thumbnail Image.png
Description
Synthetic biology and metabolic engineering has aided the production of chemicals using renewable resources, thus offering a solution to our dependence on the dwindling petroleum resources. While a major portion of petroleum resources go towards production of fuels, a significant…

Synthetic biology and metabolic engineering has aided the production of chemicals using renewable resources, thus offering a solution to our dependence on the dwindling petroleum resources. While a major portion of petroleum resources go towards production of fuels, a significant fraction also goes towards production of specialty chemicals. There has been a growing interest in recent years in commercializing bio-based production of such high value compounds. In this thesis the biosynthesis of aromatic esters has been explored, which have typical application as flavor and fragrance additive to food, drinks and cosmetics. Recent progress in pathway engineering has led to the construction of several aromatic alcohol producing pathways, the likes of which can be utilized to engineer aromatic ester biosynthesis by addition of a suitable enzyme from the acyltransferase class. Enzyme selection and screening done in this work has identified chloramphenicol O-acetyltransferase enzyme(CAT) as a potential candidate to complete the biosynthetic pathways for each of 2-phenethyl acetate, benzyl acetate, phenyl acetate and acetyl salicylate. In the end, E. coli strains capable of producing up to 60 mg/L 2-phenethyl acetate directly from glucose were successfully constructed by co-expressing CAT in a previously engineered 2-phenylethanol producing host.
Date Created
2016
Agent

Stress-responsive nano- and microcomposites featuring mechanophore units

154753-Thumbnail Image.png
Description
The problem of catastrophic damage purveys in any material application, and minimizing its occurrence is paramount for general health and safety. Thus, novel damage detection schemes are required that can sense the precursors to damage. Mechanochemistry is the area of…

The problem of catastrophic damage purveys in any material application, and minimizing its occurrence is paramount for general health and safety. Thus, novel damage detection schemes are required that can sense the precursors to damage. Mechanochemistry is the area of research that involves the use of mechanical force to induce a chemical change, with recent study focusing on directing the mechanical force to embedded mechanophore units for a targeted chemical response. Mechanophores are molecular units that provide a measureable signal in response to an applied force, often in the form of a visible color change or fluorescent emission, and their application to thermoset network polymers has been limited. Following preliminary work on polymer blends of cyclobutane-based mechanophores and epoxy, dimeric 9-anthracene carboxylic acid (Di-AC)-based mechanophore particles were synthesized and employed to form stress sensitive particle reinforced epoxy matrix composites.

Under an applied stress, the cyclooctane-rings in the Di-AC particles revert back to their fluorescent anthracene form, which linearly enhances the overall fluorescence of the composite in response to the applied strain. The fluorescent signal further allows for stress sensing in the elastic region of the stress-strain curve, which is considered to be a form of damage precursor detection. This behavior was further analyzed at the molecular scale with corresponding molecular dynamics simulations. Following the successful application of Di-AC to an epoxy matrix, the mechanophore particles were incorporated into a polyurethane matrix to show the universal nature of Di-AC as a stress-sensitive particle filler. Interestingly, in polyurethane Di-AC could successfully detect damage with less applied strain compared to the epoxy system.

While mechanophores of varying chemistries have been covalently incorporated into elastomeric and thermoplastic polymer systems, they have not yet been covalently incorporated a thermoset network polymer. Thus, following the study of mechanophore particles as stress-sensitive fillers, two routes of grafting mechanophore units into an epoxy system to form a self-sensing nanocomposite were explored. These involved the mechanophore precursor and mechanophore, cinnamamide and di-cinnamamide, respectively. With both molecules, the free amine groups can directly bond to epoxy resin to covalently incorporate themselves within the thermoset network to form a self-sensing nanocomposite.
Date Created
2016
Agent

Biosynthetic production of aromatic fine chemicals

154524-Thumbnail Image.png
Description
This dissertation focuses on the biosynthetic production of aromatic fine chemicals in engineered Escherichia coli from renewable resources. The discussed metabolic pathways take advantage of key metabolites in the shikimic acid pathway, which is responsible for the production of the…

This dissertation focuses on the biosynthetic production of aromatic fine chemicals in engineered Escherichia coli from renewable resources. The discussed metabolic pathways take advantage of key metabolites in the shikimic acid pathway, which is responsible for the production of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. For the first time, the renewable production of benzaldehyde and benzyl alcohol has been achieved in recombinant E. coli with a maximum titer of 114 mg/L of benzyl alcohol. Further strain development to knockout endogenous alcohol dehydrogenase has reduced the in vivo degradation of benzaldehyde by 9-fold, representing an improved host for the future production of benzaldehyde as a sole product. In addition, a novel alternative pathway for the production of protocatechuate (PCA) and catechol from the endogenous metabolite chorismate is demonstrated. Titers for PCA and catechol were achieved at 454 mg/L and 630 mg/L, respectively. To explore potential routes for improved aromatic product yields, an in silico model using elementary mode analysis was developed. From the model, stoichiometric optimums maximizing both product-to-substrate and biomass-to-substrate yields were discovered in a co-fed model using glycerol and D-xylose as the carbon substrates for the biosynthetic production of catechol. Overall, the work presented in this dissertation highlights contributions to the field of metabolic engineering through novel pathway design for the biosynthesis of industrially relevant aromatic fine chemicals and the use of in silico modelling to identify novel approaches to increasing aromatic product yields.
Date Created
2016
Agent

Synthesis and characterization of boronic-acid-containing metal organic frameworks

153181-Thumbnail Image.png
Description
We report the synthesis of novel boronic acid-containing metal-organic frameworks (MOFs), which was synthesized via solvothermal synthesis of cobalt nitride with 3,5-Dicarboxyphenylboronic acid (3,5-DCPBC). Powder X-ray diffraction and BET surface area analysis have been used to verify the successful synthesis…

We report the synthesis of novel boronic acid-containing metal-organic frameworks (MOFs), which was synthesized via solvothermal synthesis of cobalt nitride with 3,5-Dicarboxyphenylboronic acid (3,5-DCPBC). Powder X-ray diffraction and BET surface area analysis have been used to verify the successful synthesis of this microporous material.

We have also made the attempts of using zinc nitride and copper nitride as metal sources to synthesize the boronic acid-containing MOFs. However, the attempts were not successful. The possible reason is the existence of copper and zinc ions catalyzed the decomposition of 3,5-Dicarboxyphenylboronic acid, forming isophthalic acid. The ended product has been proved to be isophthalic acid crystals by the single crystal X-ray diffraction. The effects of solvents, reaction temperature, and added bases were investigated. The addition of triethylamine has been shown to tremendously improve the sample crystallinity by facilitating ligand deprotonation
Date Created
2014
Agent
Subscribe to Nielsen, David