Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate.

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

Genetically engineered control of phenotypic structure in microbial colonies.

Rapid advances in cellular engineering1,2 have positioned synthetic biology to address therapeutic3,4 and industrial5 problems, but a substantial obstacle is the myriad of unanticipated cellular responses in heterogeneous real-world environments such as the gut6,7, solid tumours8,9, bioreactors10 or soil11. 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 differentiation12,13. Although synthetic spatial gene expression patterns14-17 have 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.

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 ß-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.

Synthetic Gene Circuits Learn to Classify.

An efficient computational algorithm is developed to design microRNA-based synthetic cell classifiers and to optimize their performance.

Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications.

CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.

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 PL 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.

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 toward 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 reshaping 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 reshaping 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.

Structural origins of DNA target selection and nucleobase extrusion by a DNA cytosine methyltransferase.

BACKGROUND: How DNA 5-cytosine methyltransferases (DCMTases) select their substrate nucleobase for extrusion from DNA duplex is poorly understood. RESULTS: The crystal structure of a pre-extrusion M.HaeIII DCMTase-substrate DNA complex is reported here. CONCLUSION: M.HaeIII selects its substrate cytosine for extrusion by selectively interfering with its stacking and hydrogen bonding interactions within the DNA duplex. SIGNIFICANCE: This is the first structural elucidation of the target cytosine selection by a DCMTase. Epigenetic methylation of cytosine residues in DNA is an essential element of genome maintenance and function in organisms ranging from bacteria to humans. DNA 5-cytosine methyltransferase enzymes (DCMTases) catalyze cytosine methylation via reaction intermediates in which the DNA is drastically remodeled, with the target cytosine residue extruded from the DNA helix and plunged into the active site pocket of the enzyme. We have determined a crystal structure of M.HaeIII DCMTase in complex with its DNA substrate at a previously unobserved state, prior to extrusion of the target cytosine and frameshifting of the DNA recognition sequence. The structure reveals that M.HaeIII selects the target cytosine and destabilizes its base-pairing through a precise, focused, and coordinated assault on the duplex DNA, which isolates the target cytosine from its nearest neighbors and thereby facilitates its extrusion from DNA.