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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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CRISPR Based Synthetic Transcription Factors: The Future of Transcriptional Therapeutics

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Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful

Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR based transcriptional regulators have enabled researchers to perturb endogenous gene expression in vivo, allowing for the therapeutic reprogramming of cell and tissue behavior. However, for this technology to be of maximal use, a variety of technological hurdles still need to be addressed. Here, we discuss recent advances and integrative strategies that can help pave the way towards a new class of transcriptional therapeutics.

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2019-05

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Plasmid Design for Making a HEK293t Reporter Cell Line to Study Gene Expression Dynamics

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

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.

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2019-05

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

Description

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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RNA-Guided Modification of Synthetic Gene Networks Using CRISPR-Cas Systems

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The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune system against foreign nucleic acids, have recently been engineered for

The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune system against foreign nucleic acids, have recently been engineered for a plethora of genome engineering and transcriptional regulation applications. Here we employ engineered variants of CRISPR systems in proof-of-principle experiments demonstrating the ability of CRISPR-Cas derived single-DNA-strand cutting enzymes (nickases) to direct host-cell genomic recombination. E.coli is generally regarded as a poorly recombinogenic host with double-stranded DNA breaks being highly lethal. However, CRISPR-guided nickase systems can be easily programmed to make very precise, non-lethal, incisions in genomic regions directing both single reporter gene and larger-scale recombination events deleting up to 36 genes. Genome integrated repetitive elements of variable sizes can be employed as sites for CRISPR induced recombination. We project that single-stranded based editing methodologies can be employed alongside preexisting genome engineering techniques to assist and expedite metabolic engineering and minimalized genome research.

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2014-05