Matching Items (20)

128209-Thumbnail Image.png

Preparing synthetic biology for the world

Description

Synthetic Biology promises low-cost, exponentially scalable products and global health solutions in the form of self-replicating organisms, or “living devices.” As these promises are realized, proof-of-concept systems will gradually migrate

Synthetic Biology promises low-cost, exponentially scalable products and global health solutions in the form of self-replicating organisms, or “living devices.” As these promises are realized, proof-of-concept systems will gradually migrate from tightly regulated laboratory or industrial environments into private spaces as, for instance, probiotic health products, food, and even do-it-yourself bioengineered systems. What additional steps, if any, should be taken before releasing engineered self-replicating organisms into a broader user space? In this review, we explain how studies of genetically modified organisms lay groundwork for the future landscape of biosafety. Early in the design process, biological engineers are anticipating potential hazards and developing innovative tools to mitigate risk. Here, we survey lessons learned, ongoing efforts to engineer intrinsic biocontainment, and how different stakeholders in synthetic biology can act to accomplish best practices for biosafety.

Contributors

Agent

Created

Date Created
  • 2013-01-25

128013-Thumbnail Image.png

Molecular structures guide the engineering of chromatin

Description

Chromatin is a system of proteins, RNA, and DNA that interact with each other to organize and regulate genetic information within eukaryotic nuclei. Chromatin proteins carry out essential functions: packing

Chromatin is a system of proteins, RNA, and DNA that interact with each other to organize and regulate genetic information within eukaryotic nuclei. Chromatin proteins carry out essential functions: packing DNA during cell division, partitioning DNA into sub-regions within the nucleus, and controlling levels of gene expression. There is a growing interest in manipulating chromatin dynamics for applications in medicine and agriculture. Progress in this area requires the identification of design rules for the chromatin system. Here, we focus on the relationship between the physical structure and function of chromatin proteins. We discuss key research that has elucidated the intrinsic properties of chromatin proteins and how this information informs design rules for synthetic systems. Recent work demonstrates that chromatin-derived peptide motifs are portable and in some cases can be customized to alter their function. Finally, we present a workflow for fusion protein design and discuss best practices for engineering chromatin to assist scientists in advancing the field of synthetic epigenetics.

Contributors

Agent

Created

Date Created
  • 2017-06-13

Can the natural diversity of quorum-sensing advance synthetic biology?

Description

Quorum-sensing networks enable bacteria to sense and respond to chemical signals produced by neighboring bacteria. They are widespread: over 100 morphologically and genetically distinct species of eubacteria are known to

Quorum-sensing networks enable bacteria to sense and respond to chemical signals produced by neighboring bacteria. They are widespread: over 100 morphologically and genetically distinct species of eubacteria are known to use quorum sensing to control gene expression. This diversity suggests the potential to use natural protein variants to engineer parallel, input-specific, cell–cell communication pathways. However, only three distinct signaling pathways, Lux, Las, and Rhl, have been adapted for and broadly used in engineered systems. The paucity of unique quorum-sensing systems and their propensity for crosstalk limits the usefulness of our current quorum-sensing toolkit. This review discusses the need for more signaling pathways, roadblocks to using multiple pathways in parallel, and strategies for expanding the quorum-sensing toolbox for synthetic biology.

Contributors

Agent

Created

Date Created
  • 2015-03-10

127999-Thumbnail Image.png

Regulation of cancer epigenomes with a histone-binding synthetic transcription factor

Description

Chromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate

Chromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate genes by altering chromatin marks, such as histone modifications. Previously, we reported the first synthetic transcriptional activator, the “Polycomb-based transcription factor” (PcTF) that reads histone modifications through a protein–protein interaction between the polycomb chromodomain motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genome-wide behavior of the polycomb-based transcription factor fusion protein. Transcriptome and chromatin profiling revealed several polycomb-based transcription factor-sensitive promoter regions marked by distal H3K27me3 and proximal fusion protein binding. These results illuminate a mechanism in which polycomb-based transcription factor interactions bridge epigenomic marks with the transcription initiation complex at target genes. In three cancer-derived human cell lines tested here, some target genes encode developmental regulators and tumor suppressors. Thus, the polycomb-based transcription factor represents a powerful new fusion protein-based method for cancer research and treatment where silencing marks are translated into direct gene activation.

Contributors

Agent

Created

Date Created
  • 2017-01-09

135297-Thumbnail Image.png

A Synthetic Gene for Detecting Epigenetic Changes in Live Human Cells

Description

Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation

Current research into live-cell dynamics, particularly those relating to chromatin structure and remodeling, are limited. The tools that are used to detect state changes in chromatin, such as Chromatin Immunoprecipitation and qPCR, require that the cell be killed off. This limits the ability of researchers to pinpoint changes in live cells over a longer period of time. As such, there is a need for a live-cell sensor that can detect chromatin state changes. The Chromometer is a transgenic chromatin state sensor designed to better understand human cell fate and the chromatin changes that occur. HOXD11.12, a DNA sequence that attracts repressive Polycomb group (PCG) proteins, was placed upstream of a core promoter-driven fluorescent reporter (AmCyan fluorescent protein, CFP) to link chromatin repression to a CFP signal. The transgene was stably inserted at an ectopic site in U2-OS (osteosarcoma) cells. Expression of CFP should reflect the epigenetic state at the HOXD locus, where several genes are regulated by Polycomb to control cell differentiation. U2-OS cells were transfected with the transgene and grown under selective pressure. Twelve colonies were identified as having integrated parts from the transgene into their genomes. PCR testing verified 2 cell lines that contain the complete transgene. Flow cytometry indicated mono-modal and bimodal populations in all transgenic cell colonies. Further research must be done to determine the effectiveness of this device as a sensor for live cell state change detection.

Contributors

Agent

Created

Date Created
  • 2016-05

137224-Thumbnail Image.png

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

Description

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

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.

Contributors

Agent

Created

Date Created
  • 2014-05

137575-Thumbnail Image.png

Electroporation of HeLa Cells with Propidium Iodide using ""Anodisc"" Nanopore Inorganic Membrane-Buffered MEAs

Description

The use of microelectrode arrays (MEA) to electroporate cells is now a reliable way of transfecting RNA interfering substances with high viability and efficiency. However, as the 50-200 micron electrodes

The use of microelectrode arrays (MEA) to electroporate cells is now a reliable way of transfecting RNA interfering substances with high viability and efficiency. However, as the 50-200 micron electrodes are coated with many cells, there are differences in both viability and efficiency between the outside and inside of the electrode. This is due to the field created by the electrode, which has higher intensities toward the outside and lower intensities toward the middle. In order to get the electric field to spread in a more even manner, an "Anodisc" inorganic membrane seeded with cells was placed on the MEA to act as a buffer to the electric fields. One hundred percent transfection efficiency on live cells was found on one sample, though there were problems encountered along the experimental process that introduced error into the results, some of which included the inability for cells to grow to high levels of confluency on the Anodisc as well as the inverted imaging technique used on the opaque disc.

Contributors

Agent

Created

Date Created
  • 2013-05

136265-Thumbnail Image.png

ENGINEERING SYNTHETIC CHROMATIN TRANSCRIPTION FACTORS

Description

Transgene expression in mammalian cells has been shown to meet resistance in the form of silencing due to chromatin buildup within the cell. Interactions of proteins with chromatin modulate gene

Transgene expression in mammalian cells has been shown to meet resistance in the form of silencing due to chromatin buildup within the cell. Interactions of proteins with chromatin modulate gene expression profiles. Synthetic Polycomb transcription factor (PcTF) variants have the potential to reactivate these silence transgenes as shown in Haynes & Silver 2011. PcTF variants have been constructed via TypeIIS assembly to further investigate this ability to reactive transgenes. Expression in mammalian cells was confirmed via fluorescence microscopy and red fluorescent protein (RFP) expression in cell lysate. Examination of any variation in conferment of binding strength of homologous Polycomb chromodomains (PCDs) to its trimethylated lysine residue target on histone three (H3K27me3) was investigated using a thermal shift assay. Results indicate that PcTF may not be a suitable protein for surveying with SYPRO Orange, a dye that produces a detectable signal when exposed to the hydrophobic domains of the melting protein. A cell line with inducible silencing of a chemiluminescent protein was used to determine the effects PcTF variants had on gene reactivation. Results show down-regulation of the target reporter gene. We propose this may be due to PcTF not binding to its target; this would cause PcTF to deplete transcriptional machinery in the nucleus. Alternatively, the CMV promoter could be sequestering transcriptional machinery in its hyperactive transcription of PcTF leading to widespread down-regulation. Finally, the activation domain used may not be appropriate for this cell type. Future PcTF variants will address these hypotheses by including multiple Polycomb chromodomains (PCDs) to alter the binding dynamics of PcTF to its target, and by incorporating alternative promoters and activation domains.

Contributors

Agent

Created

Date Created
  • 2015-05

136133-Thumbnail Image.png

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

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.

Contributors

Created

Date Created
  • 2015-05

157715-Thumbnail Image.png

Investigating strategies to enhance microbial production of and tolerance towards aromatic biochemicals

Description

Aromatic compounds have traditionally been generated via petroleum feedstocks and have wide ranging applications in a variety of fields such as cosmetics, food, plastics, and pharmaceuticals. Substantial improvements have

Aromatic compounds have traditionally been generated via petroleum feedstocks and have wide ranging applications in a variety of fields such as cosmetics, food, plastics, and pharmaceuticals. Substantial improvements have been made to sustainably produce many aromatic chemicals from renewable sources utilizing microbes as bio-factories. By assembling and optimizing native and non-native pathways to produce natural and non-natural bioproducts, the diversity of biochemical aromatics which can be produced is constantly being improved upon. One such compound, 2-Phenylethanol (2PE), is a key molecule used in the fragrance and food industries, as well as a potential biofuel. Here, a novel, non-natural pathway was engineered in Escherichia coli and subsequently evaluated. Following strain and bioprocess optimization, accumulation of inhibitory acetate byproduct was reduced and 2PE titers approached 2 g/L – a ~2-fold increase over previously implemented pathways in E. coli. Furthermore, a recently developed mechanism to

allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.

Contributors

Agent

Created

Date Created
  • 2019