Distributed classifier based on genetically engineered bacterial cell cultures

We describe a conceptual design of a distributed classifier formed by a population of genetically engineered microbial cells. The central idea is to create a complex classifier from a population of weak or simple classifiers. We create a master population of cells with randomized synthetic biosensor circuits that have a broad range of sensitivities towards chemical signals of interest that form the input vectors subject to classification. The randomized sensitivities are achieved by constructing a library of synthetic gene circuits with randomized control sequences (e.g. ribosome-binding sites) in the front element. The training procedure consists in re-shaping of the master population in such a way that it collectively responds to the "positive" patterns of input signals by producing above-threshold output (e.g. fluorescent signal), and below-threshold output in case of the "negative" patterns. The population re-shaping is achieved by presenting sequential examples and pruning the population using either graded selection/counterselection or by fluorescence-activated cell sorting (FACS). We demonstrate the feasibility of experimental implementation of such system computationally using a realistic model of the synthetic sensing gene circuits.Comment: 31 pages, 9 figure

Bottom-up construction of complex biomolecular systems with cell-free synthetic biology

Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner

Recognizing and engineering digital-like logic gates and switches in gene regulatory networks

A central aim of synthetic biology is to build organisms that can perform useful activities in response to specified conditions. The digital computing paradigm which has proved so successful in electrical engineering is being mapped to synthetic biological systems to allow them to make such decisions. However, stochastic molecular processes have graded input-output functions, thus, bioengineers must select those with desirable characteristics and refine their transfer functions to build logic gates with digital-like switching behaviour. Recent efforts in genome mining and the development of programmable RNA-based switches, especially CRISPRi, have greatly increased the number of parts available to synthetic biologists. Improvements to the digital characteristics of these parts are required to enable robust predictable design of deeply layered logic circuits

Genetically engineered control of phenotypic structure in microbial colonies

Rapid advances in cellular engineering have positioned synthetic biology to address therapeutic and industrial problems, but a substantial obstacle is the myriad of unanticipated cellular responses in heterogeneous real-world environments such as the gut, solid tumours, bioreactors or soil. Complex interactions between the environment and cells often arise through non-uniform nutrient availability, which generates bidirectional coupling as cells both adjust to and modify their local environment through phenotypic differentiation. Although synthetic spatial gene expression patternshave been explored under homogeneous conditions, the mutual interaction of gene circuits, growth phenotype and the environment remains a challenge. Here, we design gene circuits that sense and control phenotypic structure in microcolonies containing both growing and dormant bacteria. We implement structure modulation by coupling different downstream modules to a tunable sensor that leverages Escherichia coli’s stress response and is activated on growth arrest. One is an actuator module that slows growth and thereby alters nutrient gradients. Environmental feedback in this circuit generates robust cycling between growth and dormancy in the interior of the colony, as predicted by a spatiotemporal computational model. We also use the sensor to drive an inducible gating module for selective gene expression in non-dividing cells, which allows us to radically alter population structure by eliminating the dormant phenotype with a ‘stress-gated lysis circuit‘. Our results establish a strategy to leverage and control microbial colony structure for synthetic biology applications in complex environments

Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9

The progress in development of synthetic gene circuits has been hindered by the limited repertoire of available transcription factors. Recently, it has been greatly expanded using the CRISPR/Cas9 system. However, this system is limited by its imperfect DNA sequence specificity, leading to potential crosstalk with host genome or circuit components. Furthermore, CRISPR/Cas9-mediated gene regulation is context dependent, affecting the modularity of Cas9-based transcription factors. In this paper we address the problems of specificity and modularity by developing a computational approach for selecting Cas9/gRNA transcription factor/promoter pairs that are maximally orthogonal to each other as well as to the host genome and synthetic circuit components. We validate the method by designing and experimentally testing four orthogonal promoter/repressor pairs in the context of a strong promoter P_L from phage lambda. We demonstrate that these promoters can be interfaced by constructing double and triple inverter circuits. To address the problem of modularity we propose and experimentally validate a scheme to predictably incorporate orthogonal CRISPR/Cas9 regulation into a large class of natural promoters

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze–thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury­(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics