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Translational Regulation using Artificial Introns and ncRNAs

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Synthetic biology is an emerging engineering disciple, which designs and controls biological systems for creation of materials, biosensors, biocomputing, and much more. To better control and engineer these systems, modular genetic components which allow for highly specific and high dynamic

Synthetic biology is an emerging engineering disciple, which designs and controls biological systems for creation of materials, biosensors, biocomputing, and much more. To better control and engineer these systems, modular genetic components which allow for highly specific and high dynamic range genetic regulation are necessary. Currently the field struggles to demonstrate reliable regulators which are programmable and specific, yet also allow for a high dynamic range of control. Inspired by the characteristics of the RNA toehold switch in E. coli, this project attempts utilize artificial introns and complementary trans-acting RNAs for gene regulation in a eukaryote host, S. cerevisiae. Following modification to an artificial intron, splicing control with RNA hairpins was demonstrated. Temperature shifts led to increased protein production likely due to increased splicing due to hairpin loosening. Progress is underway to demonstrate trans-acting RNA interaction to control splicing. With continued development, we hope to provide a programmable, specific, and effective means for translational gene regulation in S. cerevisae.

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2018-05

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Using Natural Diversity of Quorum Sensing to Expand the Synthetic Biology Toolbox

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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

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.

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2015-05

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Engineering cyanobacteria to convert carbon dioxide to building blocks for renewable plastics

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The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels

The production of monomer compounds for synthesizing plastics has to date been largely restricted to the petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economic feasibility. Cyanobacteria have, however, become attractive microbial factories to produce renewable fuels and chemicals directly from sunlight and CO2. To explore the feasibility of photosynthetic production of (S)- and (R)-3-hydroxybutyrate (3HB), building-block monomers for synthesizing the biodegradable plastics polyhydroxyalkanoates and precursors to fine chemicals, synthetic metabolic pathways have been constructed, characterized and optimized in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803). Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing PHB biosynthesis pathway further promoted the 3HB production. Analysis of the intracellular acetyl-CoA and anion concentrations in the culture media indicated that the phosphate consumption during the photoautotrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. Fine-tuning of the gene expression levels via strategies, including tuning gene copy numbers, promoter engineering and ribosome binding site optimization, proved critical to mitigating metabolic bottlenecks and thus improving the 3HB production. One of the engineered Synechocystis strains, namely R168, was able to produce (R)-3HB to a cumulative titer of ~1600 mg/L, with a peak daily productivity of ~200 mg/L, using light and CO2 as the sole energy and carbon sources, respectively. Additionally, in order to establish a high-efficiency transformation protocol in cyanobacterium Synechocystis 6803, methyltransferase-encoding genes were cloned and expressed to pre-methylate the exogenous DNA before Synechocystis transformation. Eventually, the transformation efficiency was increased by two orders of magnitude in Synechocystis. This research has demonstrated the use of cyanobacteria as cell factories to produce 3HB directly from light and CO2, and developed new synthetic biology tools for cyanobacteria.

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2014

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Engineering Escherichia coli for the Novel and Enhanced Biosynthesis of Phenol, Catechol, and Muconic Acid

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The engineering of microbial cell factories capable of synthesizing industrially relevant chemical building blocks is an attractive alternative to conventional petrochemical-based production methods. This work focuses on the novel and enhanced biosynthesis of phenol, catechol, and muconic acid (MA). Although

The engineering of microbial cell factories capable of synthesizing industrially relevant chemical building blocks is an attractive alternative to conventional petrochemical-based production methods. This work focuses on the novel and enhanced biosynthesis of phenol, catechol, and muconic acid (MA). Although the complete biosynthesis from glucose has been previously demonstrated for all three compounds, established production routes suffer from notable inherent limitations. Here, multiple pathways to the same three products were engineered, each incorporating unique enzyme chemistries and/or stemming from different endogenous precursors. In the case of phenol, two novel pathways were constructed and comparatively evaluated, with titers reaching as high as 377 ± 14 mg/L at a glucose yield of 35.7 ± 0.8 mg/g. In the case of catechol, three novel pathways were engineered with titers reaching 100 ± 2 mg/L. Finally, in the case of MA, four novel pathways were engineered with maximal titers reaching 819 ± 44 mg/L at a glucose yield of 40.9 ± 2.2 mg/g. Furthermore, the unique flexibility with respect to engineering multiple pathways to the same product arises in part because these compounds are common intermediates in aromatic degradation pathways. Expanding on the novel pathway engineering efforts, a synthetic ‘metabolic funnel’ was subsequently constructed for phenol and MA, wherein multiple pathways were expressed in parallel to maximize carbon flux toward the final product. Using this novel ‘funneling’ strategy, maximal phenol and MA titers exceeding 0.5 and 3 g/L, respectively, were achieved, representing the highest achievable production metrics products reported to date.

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Date Created
2017

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Engineering of Synthetic DNA/RNA Modules for Manipulating Gene Expression and Circuit Dynamics

Description

Gene circuit engineering facilitates the discovery and understanding of fundamental biology and has been widely used in various biological applications. In synthetic biology, gene circuits are often constructed by two main strategies: either monocistronic or polycistronic constructions. The Latter architecture

Gene circuit engineering facilitates the discovery and understanding of fundamental biology and has been widely used in various biological applications. In synthetic biology, gene circuits are often constructed by two main strategies: either monocistronic or polycistronic constructions. The Latter architecture can be commonly found in prokaryotes, eukaryotes, and viruses and has been largely applied in gene circuit engineering. In this work, the effect of adjacent genes and noncoding regions are systematically investigated through the construction of batteries of gene circuits in diverse scenarios. Data-driven analysis yields a protein expression metric that strongly correlates with the features of adjacent transcriptional regions (ATRs). This novel mathematical tool helps the guide for circuit construction and has the implication for the design of synthetic ATRs to tune gene expression, illustrating its potential to facilitate engineering complex gene networks. The ability to tune RNA dynamics is greatly needed for biotech applications, including therapeutics and diagnostics. Diverse methods have been developed to tune gene expression through transcriptional or translational manipulation. Control of RNA stability/degradation is often overlooked and can be the lightweight alternative to regulate protein yields. To further extend the utility of engineered ATRs to regulate gene expression, a library of RNA modules named degradation-tuning RNAs (dtRNAs) are designed with the ability to form specific 5’ secondary structures prior to RBS. These modules can modulate transcript stability while having a minimal interference on translation initiation. Optimization of their functional structural features enables gene expression level to be tuned over a wide dynamic range. These engineered dtRNAs are capable of regulating gene circuit dynamics as well as noncoding RNA levels and can be further expanded into cell-free system for gene expression control in vitro. Finally, integrating dtRNA with synthetic toehold sensor enables improved paper-based viral diagnostics, illustrating the potential of using synthetic dtRNAs for biomedical applications.

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Date Created
2020

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RNA-Based Computing Devices for Intracellular and Diagnostic Applications

Description

The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription

The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.

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Date Created
2019

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Engineering and Investigating the Effects of Renewable Chemical Production in Bacteria

Description

Metabolic engineering of bacteria has become a viable technique as a sustainable and efficient method for the production of biochemicals. Two main goals were explored: investigating styrene tolerance genes in E. coli and engineering cyanobacteria for the high yield production

Metabolic engineering of bacteria has become a viable technique as a sustainable and efficient method for the production of biochemicals. Two main goals were explored: investigating styrene tolerance genes in E. coli and engineering cyanobacteria for the high yield production of L-serine. In the first study, genes that were shown to be highly differentially expressed in E. coli upon styrene exposure were further investigated by testing the effects of their deletion and overexpression on styrene tolerance and growth. It was found that plsX, a gene responsible for the phospholipid formation in membranes, had the most promising results when overexpressed at 10 µM IPTG, with a relative OD600 of 706 ± 117% at 175 mg/L styrene when compared to the control plasmid at the same concentration. This gene is likely to be effective target when engineering styrene- and other aromatic-producing strains, increasing titers by reducing their cytotoxicity.In the second study, the goal is to engineer the cyanobacterium Synechococcus sp. PCC 7002 for the overproduction of L-serine. As a robust, photosynthetic bacteria, it has potential for being used in such-rich states to capture CO2 and produce industrially relevant products. In order to increase L-serine titers, a key degradation gene, ilvA, must be removed. While ilvA is responsible for degrading L-serine into pyruvate, it is also responsible for initiating the only known pathway for the production of isoleucine. Herein, we constructed a plasmid containing the native A0730 gene in order to investigate its potential to restore isoleucine production. If functional, a Synechococcus sp. PCC 7002 ΔilvA strain can then be engineered with minimal effects on growth and an expected increase in L-serine accumulation.

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Date Created
2021

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RNA Aptamer-Based Systems for Pathogen Detection and Biomolecule Synthesis

Description

RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two

RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two biological applications: (1) nucleic acid diagnostic assays and (2) scaffolding of enzymatic pathways. First, sensors for detecting arbitrary target RNAs based the fluorogenic RNA aptamer Broccoli are designed and validated. Studies of three different sensor designs reveal that toehold-initiated Broccoli-based aptasensors provide the lowest signal leakage and highest signal intensity in absence and in presence of the target RNA, respectively. This toehold-initiated design is used for developing aptasensors targeting pathogens. Diagnostic assays for detecting pathogen nucleic acids are implemented by integrating Broccoli-based aptasensors with isothermal amplification methods. When coupling with recombinase polymerase amplification (RPA), aptasensors enable detection of synthetic valley fever DNA down to concentrations of 2 fM. Integration of Broccoli-based aptasensors with nucleic acid sequence-based amplification (NASBA) enables as few as 120 copies/mL of synthetic dengue RNA to be detected in reactions taking less than three hours. Moreover, the aptasensor-NASBA assay successfully detects dengue RNA in clinical samples. Second, RNA scaffolds containing peptide-binding RNA aptamers are employed for programming the synthesis of nonribosomal peptides (NRPs). Using the NRP enterobactin pathway as a model, RNA scaffolds are developed to direct the assembly of the enzymes entE, entB, and entF from E. coli, along with the aryl-carrier protein dhbB from B. subtilis. These scaffolds employ X-shaped RNA motifs from bacteriophage packaging motors, kissing loop interactions from HIV, and peptide-binding RNA aptamers to position peptide-modified NRP enzymes. The resulting RNA scaffolds functionalized with different aptamers are designed and evaluated for in vitro production of enterobactin. The best RNA scaffold provides a 418% increase in enterobactin production compared with the system in absence of the RNA scaffold. Moreover, the chimeric scaffold, with E. coli and B. subtilis enzymes, reaches approximately 56% of the activity of the wild-type enzyme assembly. The studies presented in this dissertation will be helpful for future development of nucleic acid-based assays and for controlling protein interaction for NRPs biosynthesis.

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Date Created
2020