Browsing by Subject "synthetic biology"

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Antibiotic Markers: An Overlooked Design Choice in Synthetic Biology?
(2021) Young, Eleanor; Barrick, Jeffrey E.
Synthetic biology seeks to engineer living systems to enhance human health, improve material synthesis and degradation, and simplify diagnostics. When researchers design synthetic constructs, they rely on predictable host organisms and predictable genetic parts to engineer them. Antibiotics and antibiotic resistance genes are essential to synthetic designs, but different antibiotics kill cells or inhibit their growth in different ways, and antibiotic resistance genes provide resistance through different mechanisms. In general, we do not know if and how these parts impose additional fitness costs or interfere with desired genetic functions in engineered cells. To test how antibiotic choice affected synthetic devices, I examined seven near-identical plasmids. Each plasmid expressed a fluorescent protein and included a different antibiotic resistance gene. When the strains were cultured with different antibiotic concentrations, some saw a decrease in final cellular density and fluorescence while others maintained normal performance at up to ten times the selection concentration of the antibiotic. When evolved for three weeks under normal selection conditions all strains lost RCP expression by the end of the time period as populations evolved to eliminate burden. The majority of strains began to lose expression around 11 or 12 days, some lost expression as early as day 5 or as late as day 16. Each population was sequenced to reveal that the strains had accrued mutations and adjusted their copy number in different manners over the course of the experiment. Four of the strains had accumulated mutations in their RCP gene and one had mutations in the antibiotic resistant gene, some had developed possible compensatory mutations in the genome, and one of the strains with a more stable expression profile over the course of the experiment had high IS element activity and a relatively high number of mutations possibly indicating activation of the SOS response. Overall, ampicillin (beta lactamase resistance gene) was the most stable in expression while tetracycline (efflux pump resistance gene) was the least stable. These experiments show that antibiotic resistance genes, a basic design choice in synthetic biology, alter the reliability of genetic constructs.
Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis
(Mobile DNA Journal, 2014-01-13) Enyeart, Peter J.; Mohr, Georg; Ellington, Andrew D.; Lambowitz, Alan M.
Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.
A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae
(BMC System Biology, 2014-03-17) Lanza, Amanda M.; Curran, Kathleen A.; Rey, Lindsey G.; Alper, Hal S.
Background: Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results: Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms. Conclusions: Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering.
Developing a Broad-Host-Range Modular Microcin Secretion System to Protect Plants from Bacterial Pathogens
(2024) Morton, Alexa Kristine; Barrick, Jeffrey
Agricultural bacterial pathogens present a major threat to the global food supply. New methods of controlling these pathogens are badly needed, as existing methods such as applying chemical pesticides or razing contaminated crops are not sustainable. Though traditional small-molecule antibiotics have received some use in this capacity, they are hardly ideal for wide-scale application due to the ever-looming threat of antibiotic resistance. One emerging solution has been the use of biocontrol strains, beneficial bacteria that naturally kill or otherwise inhibit pathogenic strains. However, the search for new biocontrol strains relies on serendipitously finding bacteria that naturally have anti-pathogen properties. To expedite the production of new biocontrol strains, I helped develop a pipeline for engineering biocontrol strains to secrete antimicrobial peptides known as microcins. Characterized microcins have a greater specificity than other types of antibiotics, primarily targeting close phylogenetic relatives of the bacteria that naturally produce them. In order to rapidly engineer microcin-secreting biocontrol strains, I refactored a two-plasmid microcin secretion system to increase its modularity. Promoters, microcin coding sequences, signal peptides, replication origins, and even secretion systems can be interchanged in this design, allowing for an array of permutations to the system according to the user’s needs. I show that my refactored system can secrete a known microcin, inhibiting growth of its target bacterium, and I map out how broad-host-range plasmid versions of my system can be used to engineer Pseudomonas and Pantoea biocontrol strains to secrete microcins. In the future, this system can be used to test novel microcins against a multitude of pathogen targets and deploy them in diverse bacterial biocontrol chassis to protect susceptible crops.
Developing a Selection-Driven Transposition Approach for Increasing and Stabilizing Burdensome Gene Expression in Acinetobacter baylyi
(2024-04-30) Manriquez, Elizabeth; Barrick, Jeffrey E.
Transposons are mobile genetic elements that have the ability to insert themselves into the genome at locations distant from the original copy. Over generations of replication, multiple copies of genes they carry can become distributed throughout a genome. This activity could potentially be leveraged to distribute many copies of a burdensome gene within a genome, increasing its expression and reducing its risk of becoming inactivated by mutations as more copies are added to a host cell. The purpose of this study is to examine whether high and stable expression of a transgene can be accomplished via selection-driven transposition. I first engineered an Acinetobacter baylyi ADP1-ISx strain that encodes a single hyperactive Himar1 mariner transposase in its chromosome and has a cognate mini-transposon inserted at a distant location. The mini-transposon includes a selectable kanamycin resistance gene and a screenable green fluorescent protein (GFP), used as a model transgene. Adding increasing amounts of kanamycin is known to select for cells that accumulate additional copies of the kanamycin resistance gene, which can occur through transposition or tandem gene duplications. I conducted an evolution experiment over six days in which the engineered ADP1-ISx strain was serially transferred into fresh liquid media containing increasing concentrations of kanamycin. The experiment also included a negative control that consisted of the same engineered ADP1-ISx with the transposase gene removed to account for the occurrence of tandem duplications. Whole-genome sequencing of the resulting strains found that more copies of the antibiotic resistance gene evolved, but mostly due to tandem amplifications that did not result in increased expression of gfp, rather than transposition events that copied the whole mini-transposon. I also began to investigate whether engineering a new strain with higher expression of the transposase resulted in more transposition during evolution. Additionally, I studied the effects of off-target mutations in elongation factor G (FusA) that complicated these experiments. My results provide a foundation for using mini-transposons to distribute burdensome genes throughout a bacterial genome to improve their expression and stability.
Evolving microbial hosts with reduced mutation rates
(2019-05-10) McGuffy, Jenna; Barrick, Jeffrey E.
As more powerful genetic tools have been developed, metabolic engineering of microbes has expanded from the production of simple chemicals like lysine and ethanol to complex pharmaceuticals, fuels, and polymers. However, biological devices are encoded in the host’s genome and use the host’s cellular machinery; therefore, to sustain complex genetic devices, an engineered cell often uses a substantial portion of its resources, decreasing the fitness of the cell. This causes a strong selective pressure for the cell to mutate, inactivating the device and restoring the cell’s fitness. If we could reduce the mutation rate of engineered cells, it may be possible to increase the longevity of engineered biological systems, therefore improving the cells’ productivity. To evolve a strain of Saccharomyces cerevisiae with a reduced mutation rate, I performed a directed evolution experiment, Periodic Reselection for Evolutionarily Reliable Variants (PResERV) in which I periodically sorted for mutant cells that preserved burdensome fluorescent protein expression over time. After eight weeks of cell regrowth and sorting, I screened 90 mutants from five evolved populations and found that many exhibited increased maintenance of fluorescent protein expression. To determine the mutations responsible for increasing the evolutionary stability of this burdensome function, I sequenced the improved cells’ genomes. To verify that reducing the mutation rate of a strain would increase the maintenance of a complex pathway, I used a beta-carotene production assay to characterize Escherichia coli antimutator alleles discovered during a previous iteration of PResERV. Strains containing the rne antimutator allele and a combination of rne and sucD antimutator alleles maintained higher levels of beta-carotene expression compared to wildtype. The increased stability of the beta-carotene production pathway demonstrates the viability of the PResERV methodology and validates these antimutator strains for further use in synthetic biology. These experiments will provide essential knowledge about the limits to reducing mutations in common microbial hosts and a path for improving their production capabilities, so that bioengineering can be better utilized to solve global issues in agriculture and medicine.
Genetic Control of Radical Crosslinking in a Semi-Synthetic Hydrogel
(2019-09-04) Graham, Austin J.; Dundas, Christopher M.; Hillsley, Alexander Veltman; Kasprak, Dain S.; Rosales, Adrianne M.; Keitz, Benjamin K.
Enhancing materials with the qualities of living systems, including sensing, computation, and adaptation, is an important challenge in designing next-generation technologies. Living materials address this challenge by incorporating live cells as actuating components that control material function. For abiotic materials, this requires new methods that couple genetic and metabolic processes to material properties. Toward this goal, we demonstrate that extracellular electron transfer (EET) from Shewanella oneidensis can be leveraged to control radical cross-linking of a methacrylate-functionalized hyaluronic acid hydrogel. Cross-linking rates and hydrogel mechanics, specifically storage modulus, were dependent on various chemical and biological factors, including S. oneidensis genotype. Bacteria remained viable and metabolically active in the networks for a least 1 week, while cell tracking revealed that EET genes also encode control over hydrogel microstructure. Moreover, construction of an inducible gene circuit allowed transcriptional control of storage modulus and cross-linking rate via the tailored expression of a key electron transfer protein, MtrC. Finally, we quantitatively modeled hydrogel stiffness as a function of steady-state mtrC expression and generalized this result by demonstrating the strong relationship between relative gene expression and material properties. This general mechanism for radical cross-linking provides a foundation for programming the form and function of synthetic materials through genetic control over extracellular electron transfer.