Karmella A. Haynes

Preparing synthetic biology for the world

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.

Eukaryotic systems broaden the scope of synthetic biology

Synthetic biology aims to engineer novel cellular functions by assembling well-characterized molecular parts (i.e., nucleic acids and proteins) into biological “devices” that exhibit predictable behavior. Recently, efforts in eukaryotic synthetic biology have sprung from foundational work in bacteria. Designing synthetic circuits to operate reliably in the context of differentiating and morphologically complex cells presents unique challenges and opportunities for progress in the field. This review surveys recent advances in eukaryotic synthetic biology and describes how synthetic systems can be linked to natural cellular processes in order to manipulate cell behavior and to foster new discoveries in cell biology research.

Synthetic Reversal of Epigenetic Silencing

Controlling cell fate-determining gene expression is key to stem cell differentiation, tissue regeneration, and cancer therapy. To date, custom-built transcription factors recognize the information encoded in specific DNA sequences. Chromatin proteins undergo covalent modifications and form complexes that encode a second layer of information that determines proximal gene activity. Here, we employ a novel gene-targeting approach that exploits a specific chromatin modification to reactivate silenced loci in human cells. We used the human Polycomb chromatin protein and homologues from other species to construct modular synthetic transcription factors, called Pc-TFs, that recognize the repressive trimethyl-histone H3 lysine 27 (H3K27me3) signal and switch silenced genes to an active state. Pc-TF expression in U2OS osteosarcoma cells leads to increased transcription of the senescence locus CDKN2A (p16) and other loci in a chromodomain- and activation module-dependent manner, a switch to a senescence phenotype, and reduced cell proliferation. These results indicate that silenced developmental regulators can be reactivated by a synthetic transcription factor that interacts with chromatin rather than DNA, resulting in an altered cell state. As such, our work extends the flexibility of transcription factor engineering and is the first example of chromatin-mediated synthetic transcription factor targeting.

The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the systems precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.

Can the Natural Diversity of Quorum-Sensing Advance Synthetic Biology?

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.

A BioBrick compatible strategy for genetic modification of plants

BackgroundPlant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process.ResultsThis paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin.ConclusionsOur work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.

The histone deacetylase inhibitor Entinostat enhances polymer‐mediated transgene expression in cancer cell lines

Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone‐DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25‐fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer‐ mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells. Biotechnol. Bioeng. 2016;113: 1345–1356.

Molecular structures guide the engineering of chromatin

Abstract 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.

Editorial - Synthetic biology: engineering complexity and refactoring cell capabilities

Synthetic Biology is now in its second decade and many goals have been achieved toward the rational design of biological systems. This Research Topic features and reviews some of the latest progress in Synthetic Biology with a focus on research at the intersection between rational design and natural complexity with a potential outcome to concrete biotechnological applications. Kelwick et al. (2014) summarize the great expansion in the genetic toolkit and DNA assembly techniques that are currently available for synthetic biologists. These tools will advance the implementation of new functions and the production of useful metabolites in living cells in a controlled fashion. Using engineering formality, Synthetic Biology aims to identify biological design principles that can be used for practical applications. As one of the results, metabolic engineering is now becoming feasible to introduce novel functions and properties into an increasing number of microbial hosts. Examples come from Yu et al. (2014) and Heider et al. (2014) that describe the production of fatty-acid-derived chemicals and astaxanthin in microbes, respectively. Furthermore, bacteria can be engineered for the conversion of waste into renewable products, as Nieves et al. (2015) demonstrate with the bioconversion of lignocellulose.

Synthetic biology: Building genetic containment

Since the 1980s, scientists have worked on designing genetic codes to reinforce containment and control of genetically engineered microbes. New mechanistic studies of “deadman” and “passcode” gene circuits provide a flexible platform to build new safety switches.