Engineered Orthogonal Quorum Sensing Systems for Synthetic Gene Regulation in Escherichia coli

Gene regulators that are controlled by membrane-permeable compounds called homoserine lactones (HSLs) have become popular tools for building synthetic gene networks that coordinate behaviors across populations of engineered bacteria. Synthetic HSL-signaling systems are derived from natural DNA and protein elements from microbial quorum signaling pathways. Crosstalk, where a single HSL can activate multiple regulators, can lead to faults in networks composed of parallel signaling pathways. Here, we report an investigation of quorum sensing components to identify synthetic pathways that exhibit little to no crosstalk in liquid and solid cultures. In previous work, we characterized the response of a single regulator (LuxR) to 10 distinct HSL-synthase enzymes. Our current study determined the responses of five different regulators (LuxR, LasR, TraR, BjaR, and AubR) to the same set of synthases. We identified two sets of orthogonal synthase-regulator pairs (BjaI/BjaR + EsaI/TraR and LasI/LasR + EsaI/TraR) that show little to no crosstalk when they are expressed in Escherichia coli BL21. These results expand the toolbox of characterized components for engineering microbial communities

Engineering bacteria to solve the Burnt Pancake Problem

<p>Abstract</p> <p>Background</p> <p>We investigated the possibility of executing DNA-based computation in living cells by engineering <it>Escherichia coli </it>to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA.</p> <p>Results</p> <p>Inversions (or "flips") of the DNA fragment pancakes are driven by the <it>Salmonella typhimurium </it>Hin/<it>hix </it>DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in <it>E. coli</it>. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; <it>E. coli </it>cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of <it>in vivo </it>flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes.</p> <p>Conclusion</p> <p>The Hin/<it>hix </it>system is a proof-of-concept demonstration of <it>in vivo </it>computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/<it>hix </it>may provide a flexible new tool for manipulating transgenic DNA <it>in vivo</it>.</p

A BioBrick compatible strategy for genetic modification of plants

Background: Plant 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. Results: This 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. Conclusions: Our 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.Molecular and Cellular Biolog

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

The sound of silence:Transgene silencing in mammalian cell engineering

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.ISSN:2405-472

A Distinct Type of Heterochromatin Within Drosophila melanogaster Chromosome 4

Studies of transcriptional gene silencing in Drosophila melanogaster suggest that most of chromosome 4 resembles pericentric heterochromatin. However, some modifiers of position-effect variegation, including chromosome 4 dosage and loss of SU(VAR)3-9, have different effects on silencing in pericentric vs. distal arm chromosome 4 heterochromatin, distinguishing these two heterochromatin types

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