Browsing by Author "Bennett, Matthew"

Now showing 1 - 5 of 5
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    Design and Benchmarking of CRISPR/Cas Tools for Transcriptional Regulation in Human Cells
    (2023-04-20) Escobar Galdamez, Mario D; Hilton, Isaac B; Bennett, Matthew
    CRISPR/Cas systems have revolutionized biomedical research by providing easily programmable nucleic acid-targeting proteins. However, the discovery of different CRISPR systems in various organisms and their rapid proliferation in research settings have crowded the field and ultimately complicated the use of CRISPR tools. During my research, I investigated how different CRISPR/Cas9 design strategies impact the efficiency of these tools. Additionally, I quantified and compared the efficacy of CRISPR/Cas systems from a variety of different species for genome editing and transcriptional control. Finally, I leveraged these optimized design parameters to create a new synthetic system for controlling the generation of new mitochondria within living human cells.
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    Engineering genetically controlled microbial consortia
    (2016-06-24) Chen, Ye; Bennett, Matthew; Beckingham, Kathleen
    To date, the majority of synthetic gene circuits have been constructed to operate within single, isogenic cellular populations. Two of the toughest challenges for synthetic biologists to achieve complicated multi-strain systems are the limited choice of inducible signals and tuning regulatory components within a gene circuit to elicit desired outputs. Here, we describe a method that allows one to tune the dynamic range in a motif based construction of promoters with regulatory elements. To do this, we first assembled and then tested a library of promoters using different -10 and -35 sites taken from endogenous promoters within Escherichia coli. By mixing and matching the -10 and -35 sites, we were able to create a large number of ligand-inducible promoters exhibiting a wide variety of dynamic ranges. Using this method, we developed an orthogonal, tightly controlled two-signaling system. Then, we used two genetically distinct populations of Escherichia coli and this signaling system to engineer a bacterial consortium that exhibits robust oscillations in gene transcription. When co-cultured in a microfluidic device, the two strains form coupled positive and negative feedback loops at the population-level. The interacting strains exhibit robust, synchronized oscillations that are absent if either strain is cultured in isolation. We further used a combination of mathematical modeling and targeted genetic perturbations to better understand the roles of circuit topology and regulatory promoter strengths in generating and maintaining these oscillations. We found that the dual-feedback topology was robust to changes in promoter strengths and fluctuations in the population ratio of the two strains. These findings demonstrate that one can program population-level dynamics through the genetic engineering of multiple cooperative strains and point the way towards engineering complex synthetic tissues and organs with multiple cell types.
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    Examination of Stationary-Phase Gene Expression in Context of Real-World Applications of Synthetic Microbes
    (2023-04-21) Dou, Jennifer; Bennett, Matthew
    As synthetic biology attempts to move into real-world applications, it must confront the additional challenges posed by natural environments such as soil, wastewater, and the gut. Working within the gut microbiome, we sought to increase the range and potential utility of therapeutic probiotics by introducing a simple logic-gate system into the guts of mice and showing that it could work there. We were unsuccessful in this, but along the way explored issues such as what an effective reporter of gene expression that can be detected from within the mammalian gut might look like, and the additional challenges of making genetic circuitry work in stationary-phase. We developed inducible stationary-phase promoters which can be turned on by chemical inducers such as IPTG and aTc, and showed that they express primarily in stationary phase and are more effectively induced when cells are in stationary phase, compared to similar exponential-phase promoters.
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    Methods for Predicting Synthetic Gene Circuits
    (2020-02-25) Zong, David Mao; Bennett, Matthew; Ott, William
    Mature engineering disciplines use computational tools to test designs before they are built, which allows rapid engineering design-build-test cycles. Synthetic biology is an immature engineering discipline because there is a dearth of computational tools that accurately predict how engineered systems behave. A strategy to improve computational methods is to build well-defined toy systems and study them using computational modeling. In this thesis, I built two such toy systems and studied their behavior using computational modeling. The first gene circuit I designed transcribes a gene of interest in response to multiple chemical signals. This design uses modular transcription factors to increase the number of possible chemical input combinations. However, this design is difficult to fully characterize because the number of possible input concentrations and input combinations are too numerous. We constructed a predictive model that accurately predicts gene expression for a set of input chemicals at any input concentration. The next system that I built is composed of three strains of engineered E. coli that interact using cell-cell signaling. This engineered population of bacteria pulses gene expression response to external signal. This pulse was modulated by changing the population fraction of each member species. We developed a computational model that predicts the behavior of the population in response to cell strain ratio. My work shows that complex synthetic biological systems can be tuned rationally and predictably using computational tools which makes engineering biology quicker.
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    Transcriptional delay in synthetic genetic cascades
    (2017-04-19) Cheng, Yu-Yu; Phillips, George; Bennett, Matthew
    Transcription factors (TFs) and their target promoters are central to synthetic biology. By arranging these components into complex regulatory networks, synthetic biologists have been able to create a wide variety of phenotypes, including bistable switches, oscillators, and logic gates. However, transcription factors do not instantaneously regulate downstream targets. After the gene encoding a TF is turned on, it must first be transcribed, the transcripts must be translated, and sufficient TF must accumulate in order to bind operator sites of the target promoter. The time to complete this process, here called the “transcriptional delay,” is a critical aspect in the design of dynamic regulatory networks, yet it remains poorly characterized. In this work, I measured the delay of two TFs in Escherichia coli, which are commonly used in synthetic biology: the activator AraC and the repressor LacI. I found that the delay can range from a few to tens of minutes, and are affected by the expression rate of the TF. The single-cell data also shows that the variability of the delay increases with its mean. To validate these time measurements, I constructed a two-step genetic cascade, and showed that the timing of the full cascade can be predicted from those of its constituent steps. These results demonstrate the timescale of transcriptional regulation in living cells, which is important for understanding the dynamics of synthetic transcriptional gene circuits.