Modular Synthetic Inverters from Zinc Finger Proteins and Small RNAs.

Synthetic zinc finger proteins (ZFPs) can be created to target promoter DNA sequences, repressing transcription. The binding of small RNA (sRNA) to ZFP mRNA creates an ultrasensitive response to generate higher effective Hill coefficients. Here we combined three "off the shelf" ZFPs and three sRNAs to create new modular inverters in E. coli and quantify their behavior using induction fold. We found a general ordering of the effects of the ZFPs and sRNAs on induction fold that mostly held true when combining these parts. We then attempted to construct a ring oscillator using our new inverters. Our chosen parts performed insufficiently to create oscillations, but we include future directions for improvement upon our work presented here.

A genetic bistable switch utilizing nonlinear protein degradation.

BACKGROUND: Bistability is a fundamental property in engineered and natural systems, conferring the ability to switch and retain states. Synthetic bistable switches in prokaryotes have mainly utilized transcriptional components in their construction. Using both transcriptional and enzymatic components, creating a hybrid system, allows for wider bistable parameter ranges in a circuit. RESULTS: In this paper, we demonstrate a tunable family of hybrid bistable switches in E. coli using both transcriptional components and an enzymatic component. The design contains two linked positive feedback loops. The first loop utilizes the lambda repressor, CI, and the second positive feedback loop incorporates the Lon protease found in Mesoplasma florum (mf-Lon). We experimentally tested for bistable behavior in exponential growth phase, and found that our hybrid bistable switch was able to retain its state in the absence of an input signal throughout 40 cycles of cell division. We also tested the transient behavior of our switch and found that switching speeds can be tuned by changing the expression rate of mf-Lon. CONCLUSIONS: To our knowledge, this work demonstrates the first use of dynamic expression of an orthogonal and heterologous protease to tune a nonlinear protein degradation circuit. The hybrid switch is potentially a more robust and tunable topology for use in prokaryotic systems.

A feedback quenched oscillator produces turing patterning with one diffuser.

Efforts to engineer synthetic gene networks that spontaneously produce patterning in multicellular ensembles have focused on Turing's original model and the "activator-inhibitor" models of Meinhardt and Gierer. Systems based on this model are notoriously difficult to engineer. We present the first demonstration that Turing pattern formation can arise in a new family of oscillator-driven gene network topologies, specifically when a second feedback loop is introduced which quenches oscillations and incorporates a diffusible molecule. We provide an analysis of the system that predicts the range of kinetic parameters over which patterning should emerge and demonstrate the system's viability using stochastic simulations of a field of cells using realistic parameters. The primary goal of this paper is to provide a circuit architecture which can be implemented with relative ease by practitioners and which could serve as a model system for pattern generation in synthetic multicellular systems. Given the wide range of oscillatory circuits in natural systems, our system supports the tantalizing possibility that Turing pattern formation in natural multicellular systems can arise from oscillator-driven mechanisms.

BglBrick vectors and datasheets: A synthetic biology platform for gene expression.

BACKGROUND: As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors. RESULTS: Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number. CONCLUSIONS: The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.

Engineering static and dynamic control of synthetic pathways.

Maximizing the production of a desired small molecule is one of the primary goals in metabolic engineering. Recent advances in the nascent field of synthetic biology have increased the predictability of small-molecule production in engineered cells growing under constant conditions. The next frontier is to create synthetic pathways that adapt to changing environments.

Microbioreactor arrays with parametric control for high-throughput experimentation.

A scalable array technology for parametric control of high-throughput cell cultivations is demonstrated. The technology makes use of commercial printed circuit board (PCB) technology, integrated circuit sensors, and an electrochemical gas generation system. We present results for an array of eight 250 microl microbioreactors. Each bioreactor contains an independently addressable suite that provides closed-loop temperature control, generates feed gas electrochemically, and continuously monitors optical density. The PCB technology allows for the assembly of additional off-the-shelf components into the microbioreactor array; we demonstrate the use of a commercial ISFET chip to continuously monitor culture pH. The electrochemical dosing system provides a powerful paradigm for reproducible gas delivery to high-density arrays of microreactors. Growth data are presented for Escherichia coli cultured in the array with varying microaerobic conditions using electrochemically generated oxygen. Additionally, we present data on carbon dioxide generation for pH dosing.

Microbioreactor arrays with parametric control for high-throughput experimentation.

A scalable array technology for parametric control of high-throughput cell cultivations is demonstrated. The technology makes use of commercial printed circuit board (PCB) technology, integrated circuit sensors, and an electrochemical gas generation system. We present results for an array of eight 250 microl microbioreactors. Each bioreactor contains an independently addressable suite that provides closed-loop temperature control, generates feed gas electrochemically, and continuously monitors optical density. The PCB technology allows for the assembly of additional off-the-shelf components into the microbioreactor array; we demonstrate the use of a commercial ISFET chip to continuously monitor culture pH. The electrochemical dosing system provides a powerful paradigm for reproducible gas delivery to high-density arrays of microreactors. Growth data are presented for Escherichia coli cultured in the array with varying microaerobic conditions using electrochemically generated oxygen. Additionally, we present data on carbon dioxide generation for pH dosing.