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Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While

Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description
Fossil fuels have been the primary source of energy in the world for many decades. However, they are among the top contributors of the greenhouse gas emissions in the atmosphere. The objective of this research was to produce a more environmentally friendly biofuel from Algae-Helix and Salicornia biomasses. Experiments were

Fossil fuels have been the primary source of energy in the world for many decades. However, they are among the top contributors of the greenhouse gas emissions in the atmosphere. The objective of this research was to produce a more environmentally friendly biofuel from Algae-Helix and Salicornia biomasses. Experiments were conducted using a hydrothermal liquefaction (HTL) technique in the HTL reactor to produce biofuel that can potentially replace fossil fuel usage. Hydrothermal Liquefaction is a method used to convert the biomass into the biofuels. HTL experiments on Algae-Helix and Salicornia at 200°C-350°C and 430psi were performed to investigate the effect of temperature on the biocrude yield of the respective biomass used. The effect of the biomass mixture (co-liquefaction) of Salicornia and algae on the amount of biocrude produced was also explored. The biocrude and biochar (by-product) obtained from the hydrothermal liquefaction process were also analyzed using thermogravimetric analyzer (TGA). The maximum biocrude yield for the algae-helix biomass and for the Salicornia biomass were both obtained at 300°C which were 34.63% and 7.65% respectively. The co-liquefaction of the two biomasses by 50:50 provided a maximum yield of 17.26% at 250°C. The co-liquefaction of different ratios explored at 250°C and 300°C concluded that Salicornia to algae-helix ratio of 20:80 produced the highest yields of 22.70% and 31.97%. These results showed that co-liquefaction of biomass if paired well with the optimizing temperature can produce a high biocrude yield. The TGA profiles investigated have shown that salicornia has higher levels of ash content in comparison with the algae-helix. It was then recommended that for a mixture of algae and Salicornia, large-scale biofuel production should be conducted at 250℃ in a 20:80 salicornia to algae biocrude ratio, since it lowers energy needs. The high biochar content left can be recycled to optimize biomass, and prevent wastage.
ContributorsLaideson, Maymary Everrest (Co-author) / Luboowa, Kato (Co-author) / Deng, Shuguang (Thesis director) / Nielsen, David (Committee member) / Chemical Engineering Program (Contributor) / Economics Program in CLAS (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Fossil fuels have been the primary source of energy in the world for many decades. However, they are among the top contributors of the greenhouse gas emissions in the atmosphere. The objective of this research was to produce a more environmentally friendly biofuel from Algae-Helix and Salicornia biomasses. Experiments

Fossil fuels have been the primary source of energy in the world for many decades. However, they are among the top contributors of the greenhouse gas emissions in the atmosphere. The objective of this research was to produce a more environmentally friendly biofuel from Algae-Helix and Salicornia biomasses. Experiments were conducted using a hydrothermal liquefaction (HTL) technique in the HTL reactor to produce biofuel that can potentially replace fossil fuel usage. Hydrothermal Liquefaction is a method used to convert the biomass into the biofuels. HTL experiments on Algae-Helix and Salicornia at 200°C-350°C and 430psi were performed to investigate the effect of temperature on the biocrude yield of the respective biomass used. The effect of the biomass mixture (co-liquefaction) of Salicornia and algae on the amount of biocrude produced was also explored. The biocrude and biochar (by-product) obtained from the hydrothermal liquefaction process were also analyzed using thermogravimetric analyzer (TGA). The maximum biocrude yield for the algae-helix biomass and for the Salicornia biomass were both obtained at 300°C which were 34.63% and 7.65% respectively. The co-liquefaction of the two biomasses by 50:50 provided a maximum yield of 17.26% at 250°C. The co-liquefaction of different ratios explored at 250°C and 300°C concluded that Salicornia to algae-helix ratio of 20:80 produced the highest yields of 22.70% and 31.97%. These results showed that co-liquefaction of biomass if paired well with the optimizing temperature can produce a high biocrude yield. The TGA profiles investigated have shown that salicornia has higher levels of ash content in comparison with the algae-helix. It was then recommended that for a mixture of algae and Salicornia, large-scale biofuel production should be conducted at 250℃ in a 20:80 salicornia to algae biocrude ratio, since it lowers energy needs. The high biochar content left can be recycled to optimize biomass, and prevent wastage.
ContributorsLuboowa, Kato Muhammed (Co-author) / Laideson, Maymary (Co-author) / Deng, Shuguang (Thesis director) / Nielsen, David (Committee member) / Chemical Engineering Program (Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Due to the wide range of health properties flavonoids possess, flavonoids are sold in health supplements to the general public. Flavonoids are also utilized in research but have a high cost due to current production techniques. This project focuses on engineering two DNA recombinants to develop new strains of Corynebacterium

Due to the wide range of health properties flavonoids possess, flavonoids are sold in health supplements to the general public. Flavonoids are also utilized in research but have a high cost due to current production techniques. This project focuses on engineering two DNA recombinants to develop new strains of Corynebacterium glutamicum that can produce flavonoids pinocembrin and naringenin. After culturing Escherichia coli colonies containing genes of interest, the genes were collected and purified by PCR reactions. The recombinant plasmid was assembled using CPEC and successfully transformed into Escherichia coli, with plans to transform Corynebacterium glutamicum to experiment and determine which recombinant can produce more pinocembrin and naringenin. Design work for other DNA recombinants, which were not the focus of this project, was also completed.
ContributorsWong, Adam (Co-author, Co-author) / Varman, Arul Mozhy (Thesis director) / Nielsen, David (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Cyanobacteria have the potential to efficiently produce L-serine, an industrially important amino acid, directly from CO2 and sunlight, which is a more sustainable and inexpensive source of energy as compared to current methods. The research aims to engineer a strain of Cyanobacterium Synechococcus sp. PCC 7002 that increases L-serine production

Cyanobacteria have the potential to efficiently produce L-serine, an industrially important amino acid, directly from CO2 and sunlight, which is a more sustainable and inexpensive source of energy as compared to current methods. The research aims to engineer a strain of Cyanobacterium Synechococcus sp. PCC 7002 that increases L-serine production by mutating regulatory mechanisms that natively inhibit its production and encoding an exporter. While an excess of L-serine was not found in the supernatant of the cell cultures, with further fine tuning of the metabolic pathway and culture conditions, high titers of L-serine can be found. With the base strain engineered, the work can be extended and optimized by deleting degradation pathways, tuning gene expression levels, optimizing growth conditions, and investigating the effects of nitrogen supplementation for the strain.
ContributorsAbed, Omar (Author) / Nielsen, David (Thesis director) / Jones, Christopher (Committee member) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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Description
The field of bioprocess engineering has become an increasingly popular route to produce chemicals and fuels in a sustainable fashion. Bioprocessing is an interdisciplinary field that joins chemical engineering, metabolic engineering, and synthetic biology to tackle problems that will arise from the ongoing use of products derived from non-renewable resources.

The field of bioprocess engineering has become an increasingly popular route to produce chemicals and fuels in a sustainable fashion. Bioprocessing is an interdisciplinary field that joins chemical engineering, metabolic engineering, and synthetic biology to tackle problems that will arise from the ongoing use of products derived from non-renewable resources. This study will overlook two effective tools that are widely used in the bioprocessing field. The first tool that was studied was strain optimization for biochemical production. This involves genetic manipulation of microbial hosts to create strains that are more efficient at producing the desired products. The second tool that was studied was adaptive laboratory evolution which is used to enhance overall cellular fitness. Enhancing the overall fitness and efficiency of these microbial production factories, allows for innovation and growth in the biochemical industry. Creating sustainable and renewable solutions for both specialty and commodity chemicals.
Strain optimization was specifically studied by enhancing inorganic carbon uptake in synechococcus sp. 7002. It is desired to have both high flux and high affinity transport for the rapid and efficient uptake of HCO3- for enhanced cell growth. The results found that the regulatory gene for carbon transporters in synechococcus genome was successfully deleted. Increasing the toxicity limits of 2-Phenylethanol was done by using adaptive laboratory evolution (ALE). ALE is a widely used practice in biotechnology studies to gain insights on mechanisms of molecular evolution and to better define the functionality of microbial cell factories. It was found that after growing E. coli BW25113 under selective conditions the genome evolved for a higher fitness medium with an increased concentration of 2-Phenylethanol. Overall, two key tools used in bioprocess engineering were successful studied to gain a better insight on the future of biochemical production industry.
ContributorsSteeber, Gavin Lyle (Author) / Nielsen, David (Thesis director) / Jones, Christopher (Committee member) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and

Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and differentiation has proven challenging. A better understanding of cell differentiation has applications in regenerative stem cell therapies, disease pathologies, and gene regulatory networks.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
ContributorsLoveday, Tristan Andre (Author) / Wang, Xiao (Thesis director) / Brafman, David (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E.

Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E. coli fermentative growth being less robust compared to some microbes such as Lactobacilli under anaerobic and microaerobic fermentation conditions. Identification and characterization of its fermentative growth constraints will help in making E. coli a better fermentation host. In this thesis, I demonstrate that Lactobacillus plantarum WCFS1 has desirable fermentative capabilities that may be transferrable to E. coli through genetic engineering to alleviate growth restraints. This has led to the hypothesis that these L. plantarum DNA sequences are transferrable through a genomic library. A background of comparative genomics and complementary literature review has demonstrated that E. coli growth may be hindered by stress from many toxin-antitoxin systems. L. plantarum WCFS1 optimizes amino acid catabolism over glycolysis to generate high ATP levels from reducing agents and proton motive force, and Lactobacilli are resistant to acidic environments and encodes a wide variety of acid transporters that could help E. coli fermentative growth. Since a great variety of L. plantarum genes may contribute to its fermentative capabilities, a gDNA library containing L. plantarum WCFS1 genes has been successfully constructed for testing in E. coli bioproducers to search for specific genes that may enhance E. coli fermentative performance and elucidate the molecular basis of Lactobacillus fermentative success.
ContributorsDufault, Matthew Elijah (Co-author, Co-author) / Wang, Xuan (Thesis director) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and L-Phenylalanine—a precursor to many pharmaceuticals. Significant cell growth occurs in

Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and L-Phenylalanine—a precursor to many pharmaceuticals. Significant cell growth occurs in parallel to the biosynthesis of the desired biofuel or biochemical product, and limits product concentrations and yields. Stopping cell growth can improve chemical production since more resources will go toward chemical production than toward biomass. The goal of the project is to test different methods of controlling microbial uptake of nutrients, specifically phosphate, to dynamically limit cell growth and improve biochemical production of E. coli, and the research has the potential to promote public health, sustainability, and environment. This can be achieved by targeting phosphate transporter genes using CRISPRi and CRISPR, and they will limit the uptake of phosphate by targeting the phosphate transporter genes in DNA, which will stop transcriptions of the genes. In the experiment, NST74∆crr∆pykAF, a L-Phe overproducer, was used as the base strain, and the pitA phosphate transporter gene was targeted in the CRISPRi and CRISPR systems with the strain with other phosphate transporters knocked out. The tested CRISPRi and CRISPR mechanisms did not stop cell growth or improved L-Phe production. Further research will be conducted to determine the problem of the system. In addition, the CRISPRi and CRISPR systems that target multiple phosphate transporter genes will be tested in the future as well as the other method of stopping transcriptions of the phosphate transporter genes, which is called a tunable toggle switch mechanism.
ContributorsPark, Min Su (Author) / Nielsen, David (Thesis director) / Machas, Michael (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain

One of the primary bottlenecks to chemical production in biological organisms is the toxicity of the chemical. Overexpression of efflux pumps has been shown to increase tolerance to aromatic compounds such as styrene and styrene oxide. Tight control of pump expression is necessary to maximize titers and prevent excessive strain on the cells. This study aimed to identify aromatic-sensitive native promoters and heterologous biosensors for construction of closed-loop control of efflux pump expression in E. coli. Using a promoter library constructed by Zaslaver et al., activation was measured through GFP output. Promoters were evaluated for their sensitivity to the addition of one of four aromatic compounds, their "leaking" of signal, and their induction threshold. Out of 43 targeted promoters, 4 promoters (cmr, mdtG, yahN, yajR) for styrene oxide, 2 promoters (mdtG, yahN) for styrene, 0 promoters for 2-phenylethanol, and 1 promoter for phenol (pheP) were identified as ideal control elements in aromatic bioproduction. In addition, a series of three biosensors (NahR, XylS, DmpR) known to be inducible by other aromatics were screened against styrene oxide, 2-phenylethanol, and phenol. The targeted application of these biosensors is aromatic-induced activation of linked efflux pumps. All three biosensors responded strongly in the presence of styrene oxide and 2-phenylethanol, with minor activation in the presence of phenol. Bioproduction of aromatics continues to gain traction in the biotechnology industry, and the continued discovery of aromatic-inducible elements will be essential to effective pathway control.
ContributorsXu, Jimmy (Author) / Nielsen, David (Thesis director) / Wang, Xuan (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05