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bioRxiv2015

Generating effective models and parameters for RNA genetic circuits

RNA genetic circuitry is emerging as a powerful tool to control gene expression. However, little work has been done to create a theoretical foundation for RNA circuit design. A prerequisite to this is a quantitative modeling framework that accurately describes the dynamics of RNA circuits. In this work, we develop an ordinary differential equation model of transcriptional RNA genetic circuitry, using an RNA cascade as a test case. We show that parameter sensitivity analysis can be used to design a set of four simple experiments that can be performed in parallel using rapid cell-free transcription-translation (TX-TL) reactions to determine the thirteen parameters of the model. The resulting model accurately recapitulates the dynamic behavior of the cascade, and can be easily extended to predict the function of new cascade variants that utilize new elements with limited additional characterization experiments. Interestingly, we show that inconsistencies between model predictions and experiments led to the model-guided discovery of a previously unknown maturation step required for RNA regulator function. We also determine circuit parameters in two different batches of TX-TL, and show that batch-to-batch variation can be attributed to differences in parameters that are directly related to the concentrations of core gene expression machinery. We anticipate the RNA circuit models developed here will inform the creation of computer aided genetic circuit design tools that can incorporate the growing number of RNA regulators, and that the parameterization method will find use in determining functional parameters of a broad array of natural and synthetic regulatory systems.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC=\"FIGDIR/small/018358_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (17K):\norg.highwire.dtl.DTLVardef@1eace4dorg.highwire.dtl.DTLVardef@8e2757org.highwire.dtl.DTLVardef@b8f454org.highwire.dtl.DTLVardef@b0cfc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Synthetic Biology↗

bioRxiv2015

Protein degradation in a TX-TL cell-free expression system using ClpXP protease

Introduction Introduction RESULTS AND DISCUSSION CONCLUSION ACKNOWLEDGEMENTS MATERIALS AND METHODS REFERENCES An in vitro S30-based Escherichia coli expression system (\"Transcription-Translation\", or \"TX-TL\") has been developed as an alternative prototyping environment to the cell for synthetic circuits [1-5]. Basic circuit elements, such as switches and cascades, have been shown to function in TX-TL, as well as bacteriophage assembly [2, 6]. Circuits can also be prototyped from basic parts within 8 hours, avoiding cloning and transformation steps [7]. However, most published results have been obtained in a \"batch mode\" reaction, where factors that play an important role for in vivo circuit dynamics ...

Synthetic Biology↗

bioRxiv2015

Cell-cell communication in yeast using auxin biosynthesis and auxin responsive CRISPR transcription factors

A true engineering framework for synthetic multicellular systems requires a programmable means of cell-cell communication. Such a communication system would enable complex behaviors, such as pattern formation, division of labor in synthetic microbial communities, and improved modularity in synthetic circuits. However, it remains challenging to build synthetic cellular communication systems in eukaryotes due to a lack of molecular modules that are orthogonal to the host machinery, easy to reconfigure, and scalable. Here, we present a novel cell-to-cell communication system in Saccharomyces cerevisiae (yeast) based on CRISPR transcription factors and the plant hormone auxin that exhibits several of these features. Specifically, we engineered a sender strain of yeast that converts indole-3-acetamide (IAM) into auxin via the enzyme iaaH from Agrobacterium tumefaciens. To sense auxin and regulate transcription in a receiver strain, we engineered a reconfigurable library of auxin degradable CRISPR transcription factors (ADCTFs). Auxin-induced degradation is achieved through fusion of an auxin sensitive degron (from IAA co-repressors) to the CRISPR TF and co-expression with an auxin F-box protein. Mirroring the tunability of auxin perception in plants, our family of ADCTFs exhibits a broad range of auxin sensitivities. We characterized the kinetics and steady state behavior of the sender and receiver independently, and in co-cultures where both cell types were exposed to IAM. In the presence of IAM, auxin is produced by the sender cell and triggers de-activation of reporter expression in the receiver cell. The result is an orthogonal, rewireable, tunable, and arguably scalable cell-cell communication system for yeast and other eukaryotic cells.

Synthetic Biology↗

bioRxiv2015

Complete absence of thebaine biosynthesis under home-brew fermentation conditions

Yeast-based biosynthesis of medicinal compounds traditionally derived from plant materials is improving. Both concerns and hopes exist for the possibility that individual small volume batch fermentations could provide distributed and independent access to a diversity of compounds some of which are now abused, illegal, or unavailable to many who need for genuine medical purposes. However, there are differences between industrial bioreactors and home-brew fermentation. We used engineered yeast that make thebaine, a morphinan opiate, to quantify if differences in fermentation conditions impact biosynthesis yields. We used yeast that make an English ale as a positive fermentation control. We observed no production of thebaine and miniscule amounts of reticuline, an upstream biosynthetic intermediate, in home-brew fermentations; the positive control was palatable. We suggest that additional technical challenges, some of which are unknown and likely unrelated to optimized production in large-volume bioreactors, would need to be addressed for engineered yeast to ever realize home-brew biosynthesis of medicinal opiates at meaningful yields.

Synthetic Biology↗

bioRxiv2015

Design Space Exploration of the Violacein Pathway in Escherichia coli Based Transcription Translation Cell-Free System (TX-TL)

In this study, an Escherichia coli (E. coli) based transcription translation cell-free system (TX-TL) was employed to sample various enzyme expression levels of the violacein pathway. The pathway was successfully reconstructed in TX-TL. Its variation produced different metabolites as evident from the extracts assorted colors. Analysis of the violacein product via UV-Vis absorption and liquid chromatography-mass spectrometry (LC-MS) detected 68 ng of violacein per l reaction volume. Significant buildup of prodeoxyviolacein intermediate was also detected in the equimolar TX-TL reaction. Finally, design space exploration experiments suggested an improvement in violacein production at high VioC and VioD DNA concentrations.

Synthetic Biology↗

bioRxiv2015

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER), a novel method for selective transformation

The CRISPR/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be applied to facilitate efficient plasmid selection for transformation as well as selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts after restriction enzyme digestion and ligation. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes unwanted ligation byproducts and increases transformation efficiency of desired inserts from 20% up to 97% {+/-} 3%. This CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient method for obtaining specific cloning products.

Synthetic Biology↗

bioRxiv2015

A synthetic gene circuit for measuring autoregulatory feedback control.

Auto regulatory feedback loops occur in the regulation of molecules ranging from ATP to MAP kinases to zinc. Negative feedback loops can increase a systems robustness, while positive feedback loops can mediate transitions between cell states. Recent genome-wide experimental and computational studies predict hundreds of novel feedback loops. However, not all physical interactions are regulatory, and many experimental methods cannot detect self-interactions. Our understanding of regulatory feedback loops is therefore hampered by the lack of high-throughput methods to experimentally quantify the presence, strength, and temporal dynamics of auto regulatory feedback loops. Here we present a mathematical and experimental framework for high-throughput quantification of feedback regulation, and apply it to RNA binding proteins (RBPs) in yeast. Our method is able to determine the existence of both direct and indirect positive and negative feedback loops, and to quantify the strength of these loops. We experimentally validate our model using two RBPs which lack native feedback loops, and by the introduction of synthetic feedback loops. We find that the the RBP Puf3 does not natively participate in any direct or indirect feedback regulation, but that replacing the native 3UTR with that of COX17 generates an auto-regulatory negative feedback loop which reduces gene expression noise. Likewise, the RBP Pub1 does not natively participate in any feedback loops, but a synthetic positive feedback loop involving Pub1 results in increased expression noise. Our results demonstrate a synthetic experimental system for quantifying the existence and strength of feedback loops using a combination of high-throughput experiments and mathematical modeling. This system will be of great use in measuring auto-regulatory feedback by RNA binding proteins, a regulatory motif that is difficult to quantify using existing high-throughput methods.

Synthetic Biology↗

bioRxiv2015

A population-based temporal logic gate for timing and recording chemical events

Single-cell bacterial sensors have numerous applications in human health monitoring, environmental chemical detection, and materials biosynthesis. Such bacterial devices need not only the capability to differentiate between combinations of inputs, but also the ability to process signal timing and duration. In this work, we present a two-input temporal logic gate that can sense and record the order of the inputs, the timing between inputs, and the duration of input pulses. The temporal logic gate design relies on unidirectional DNA recombination with bacteriophage integrases to detect and encode sequences of input events. When implemented in a chromosomally-modified E. coli strain, we can utilize stochastic single cell responses to predict overall heterogeneous population behavior. We show that a stochastic model can be used to predict final population distributions of this E. coli strain, and thus that final differentiated sub-populations can be used to deduce the timing and duration of transient chemical events.

Synthetic Biology↗

bioRxiv2016

When Wavelengths Collide: Bias in Cell Abundance Measurements due to Expressed Fluorescent Proteins

The abundance of bacteria in liquid culture is commonly inferred by measuring optical density at 600 nm. Red fluorescent proteins (RFP) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%. Measuring optical density at 700 nm would allow estimation of cell abundance unaffected by the presence of nearly all fluorescent proteins.

Synthetic Biology↗

bioRxiv2016

Robust digital logic circuits in eukaryotic cells with CRISPR/dCas9 NOR gates

Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used genetic components leak transcriptionally, do not allow arbitrary interconnections, or do not have digital responses. Here, we designed a new dCas9-Mxi1 based NOR gate architecture in S. cerevisiae that allows arbitrary connectivity and large genetic circuits. Because we used the strong chromatin remodeler Mxi1, our system showed very little leak and exhibits a highly digital response. In particular, we built a combinatorial library of NOR gates that each directly convert guide RNA (gRNA) input signals into gRNA output signals, enabling NOR gates to be \"wired\" together. We constructed and characterized logic circuits with up to seven independent gRNAs, including repression cascades with up to seven layers. Modeling predicted that the NOR gates have Hill Coefficients of approximately 1.71 {+/-} 0.09, explaining the minimal signal degradation we observed in these deeply layered circuits. Our approach enables the construction of the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, decision making systems in living cells.

Synthetic Biology↗

bioRxiv2016

Gene cloning and construction of prokaryotic and plant expression vectors of RICIN-A-Chain/PAP-S1 fusion protein and its inhibition of protein synthesis

INTRODUCTION INTRODUCTION MATERIALS AND METHODS Design of fusion DNA... DNA Sequencing RESULTS AND DISCUSSION Conclusion References Plants produce toxic proteins known as ribosome inactivating proteins (RIPs) essential to their defense mechanism against outside pathogens. RIPs are RNA N-glycosidases that function by irreversibly inhibiting protein synthesis through the removal of one or more adenine residues from ribosomal RNA (rRNA) [1]. In addition, certain RIPs can remove adenine from DNA and other polynucleotides for which reason they are also known as polynucleotide adenosine glycosidases. Pokeweed antiviral protein (PAP), a RIP from Phytolacca americana, can cleave not only adenine, but also guanine from the rRNA of Escherichia coli. RIPs are usually categ ...

Synthetic Biology↗

bioRxiv2016

Evolutionary dynamics of CRISPR gene drives

The alteration of wild populations has been discussed as a solution to a number of humanitys most pressing ecological and public health concerns. Enabled by the recent revolution in genome editing, CRISPR gene drives, selfish genetic elements which can spread through populations even if they confer no advantage to their host organism, are rapidly emerging as the most promising approach. But before real-world applications are considered, it is imperative to develop a clear understanding of the outcomes of drive release in nature. Toward this aim, we mathematically study the evolutionary dynamics of CRISPR gene drives. We demonstrate that the emergence of drive-resistant alleles presents a major challenge to previously reported constructs, and we show that an alternative design which selects against resistant alleles greatly improves evolutionary stability. We discuss all results in the context of CRISPR technology and provide insights which inform the engineering of practical gene drive systems.

Synthetic Biology↗

bioRxiv2016

Daisy-chain gene drives for the alteration of local populations

RNA-guided gene drive elements could address many ecological problems by altering the traits of wild organisms, but the likelihood of global spread tremendously complicates ethical development and use. Here we detail a localized form of CRISPR-based gene drive composed of genetic elements arranged in a daisy-chain such that each element drives the next. \"Daisy drive\" systems can duplicate any effect achievable using an equivalent global drive system, but their capacity to spread is limited by the successive loss of non-driving elements from the base of the chain. Releasing daisy drive organisms constituting a small fraction of the local wild population can drive a useful genetic element to local fixation for a wide range of fitness parameters without resulting in global spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionary stable daisy drive as well as global CRISPR-based gene drive. Daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.\n\nAuthors Summary Global gene drive systems based on CRISPR are likely to spread to every population of the target species, hampering safe and ethical use. Daisy drive systems offer a way to alter the traits of only local populations in a temporary manner. Because they can exactly duplicate the activity of any global CRISPR-based drive at a local level, daisy drives may enable safe field trials and empower local communities to make decisions concerning their own shared environments.\n\nFor more details and an animation intended for a general audience, see the summary at Sculpting Evolution.

Synthetic Biology↗

bioRxiv2016

Modular non-repeating codes for DNA storage

1We describe a strategy for constructing codes for DNA-based information storage by serial composition of weighted finite-state transducers. The resulting state machines can integrate correction of substitution errors; synchronization by interleaving watermark and periodic marker signals; conversion from binary to ternary, quaternary or mixed-radix sequences via an efficient block code; encoding into a DNA sequence that avoids homopolymer, dinucleotide, or trinucleotide runs and other short local repeats; and detection/correction of errors (including local duplications, burst deletions, and substitutions) that are characteristic of DNA sequencing technologies. We present software implementing these codes, available at https://github.com/ihh/dnastore, with simulation results demonstrating that the generated DNA is free of short repeats and can be accurately decoded even in the presence of substitutions, short duplications and deletions.

Synthetic Biology↗

bioRxiv2016

Characterizing the Non-Normal Distribution of Flow Cytometry Measurements from Transiently Expressed Constructs in Mammalian Cells

In mammalian cells, transient gene expression (TGE) is a rapid, minimal-investment alternative to single-copy integrations for testing of transgenic constructs. However, transient gene expression, as measured by flow cytometry with a fluorescent reporter, typically displays a broad, asymmetric distribution with a left-tail that is convolved with background signal. Common approaches for deriving a summary statistic for transiently expressed gene products impose a normal distribution on gated or ungated data. Summary statistics derived from these models are heavily biased by experimental conditions and instrument settings that are difficult to replicate and insufficient to accurately describe the underlying data. Here, we present a convolved gamma distribution as a superior model for TGE datasets. The 4-6 parameters of this model are sufficient to accurately describe the entire, ungated distribution of transiently transfected HEK cells expressing monomeric fluorescent proteins, that operates consistently across a range of transfection conditions and instrument settings. Based on these observations, a convolved gamma model of TGE distributions has the potential to significantly improve the accuracy and reproducibility of genetic device characterization in mammalian cells.

Synthetic Biology↗

bioRxiv2016

Precise Cas9 targeting enables genomic mutation prevention

Here we present a generalized method of guide RNA \"tuning\" that enables Cas9 to discriminate between two target sites that differ by a single nucleotide polymorphism. We employ our methodology to generate a novel in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the designed mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

Synthetic Biology↗

bioRxiv2016

The role of single occupancy effects on integrase dynamics in a cell-free system

Phage integrase-based circuits are an alternative approach to relying on transcriptional and translational repression for biomolecular circuits. Previous research has shown that circuits based on integrases can perform a variety of functions, including counters, Boolean logic operators, memory modules and temporal event detectors. It is therefore essential to develop a principled theoretical and experimental framework for the design, implementation and study of such circuits. One of the fundamental questions that such a framework should address concerns the functionality limitations and temporal dynamics of the integrases as regulatory elements. Here, we test the functionality of several large serine integrases from a recently published library in a cell-free transcription-translation (TX-TL) platform. Additionally, we use a combination of experimental data and models to investigate integrase dynamics as a function of enzyme concentration and number of binding sites.\n\nWe report that sequestration of integrase molecules, either in the form of monomers or dimers, by the integrase's own binding sites dominates integrase dynamics, and that the delay in the activation of the reporter is negatively correlated with integrase plasmid concentration. We have validated our sequestration hypothesis by building a model with MATLABs SimBiology toolbox, and running simulations with various integrase and binding sites concentrations. The simulation results qualitatively match the experimental results, and offer further insights into the system.

Synthetic Biology↗

bioRxiv2016

Synthetic Photosynthetic Consortia Define Interactions Leading to Robustness and Photoproduction

BackgroundMicrobial consortia composed of autotrophic and heterotrophic species abound in nature, yet examples of synthetic communities with mixed metabolism are limited in the laboratory. We previously engineered a model cyanobacterium, Synechococcus elongatus PCC 7942, to secrete the bulk of the carbon it fixes as sucrose, a carbohydrate that can be utilized by many other microbes. Here, we tested the capability of sucrose-secreting cyanobacteria to act as a flexible platform for the construction of synthetic, light-driven consortia by pairing them with three disparate heterotrophs: Bacillus subtilis, Escherichia coli, or Saccharomyces cerevisiae. The comparison of these different co-culture dyads reveals general design principles for the construction of robust autotroph/heterotroph consortia.\n\nMain findingsWe observed heterotrophic growth dependent upon cyanobacterial photosynthate in each co-culture pair. Furthermore, these synthetic consortia could be stabilized over the long-term (weeks to months) and both species could persist when challenged with specific perturbations. Stability and productivity of autotroph/heterotroph co-cultures was dependent on heterotroph sucrose utilization, as well as other species-independent interactions that we observed across all dyads. One interaction we observed to destabilize consortia was that non-sucrose byproducts of photosynthesis negatively impacted heterotroph growth. Conversely, inoculation of each heterotrophic species enhanced cyanobacterial growth in comparison to axenic cultures Finally, these consortia can be flexibly programmed for photoproduction of target compounds and proteins; by changing the heterotroph in co-culture to specialized strains of B. subtilis or E. coli we demonstrate production of alpha-amylase and polyhydroxybutyrate, respectively.\n\nConclusionsEnabled by the unprecedented flexibility of this consortia design, we uncover species-independent design principles that influence cyanobacteria/heterotroph consortia robustness. The modular nature of these communities and their unusual robustness exhibits promise as a platform for highly-versatile photoproduction strategies that capitalize on multi-species interactions and could be utilized as a tool for the study of nascent symbioses. Further consortia improvements via engineered interventions beyond those we show here (i.e. increased efficiency growing on sucrose) could improve these communities as production platforms.

Synthetic Biology↗