Characterizing chemical signaling between engineered "microbial sentinels" in porous microplates.

Living materials combine a material scaffold, that is often porous, with engineered cells that perform sensing, computing, and biosynthetic tasks. Designing such systems is difficult because little is known regarding signaling transport parameters in the material. Here, the development of a porous microplate is presented. Hydrogel barriers between wells have a porosity of 60% and a tortuosity factor of 1.6, allowing molecular diffusion between wells. The permeability of dyes, antibiotics, inducers, and quorum signals between wells were characterized. A "sentinel" strain was constructed by introducing orthogonal sensors into the genome of Escherichia coli MG1655 for IPTG, anhydrotetracycline, L-arabinose, and four quorum signals. The strain's response to inducer diffusion through the wells was quantified up to 14 mm, and quorum and antibacterial signaling were measured over 16 h. Signaling distance is dictated by hydrogel adsorption, quantified using a linear finite element model that yields adsorption coefficients from 0 to 0.1 mol m-3 . Parameters derived herein will aid the design of living materials for pathogen remediation, computation, and self-organizing biofilms.

Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors.

Cellular processes are carried out by many genes, and their study and optimization requires multiple levers by which they can be independently controlled. The most common method is via a genetically encoded sensor that responds to a small molecule. However, these sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we have developed a directed evolution strategy to simultaneously select for lower background, high dynamic range, increased sensitivity, and low cross-talk. This is applied to generate a set of 12 high-performance sensors that exhibit >100-fold induction with low background and cross-reactivity. These are combined to build a single "sensor array" in the genomes of E. coli MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These "Marionette" strains allow for the independent control of gene expression using 12 small-molecule inducers.

Transcription yield of fully 2'-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants.

On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed.

A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase.

Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa 'σ fragment', and fragments with different specificity can direct the remaining 601-aa 'core fragment' to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal 'α fragment' and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids.

Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome-nucleus association and transcriptional signaling.

The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments. Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic proteins NuMA and LAP2α also have aberrant distributions, but lamin B and LAP2ß have normal localizations. 4.1R-deficient mouse embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the association of 4.1R with emerin and A-type lamin: increased nucleus-centrosome distances, increased ß-catenin signaling, and relocalization of ß-catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact upon nuclear architecture, centrosome-nuclear envelope association and the regulation of ß-catenin transcriptional co-activator activity that is dependent on ß-catenin nuclear export.

Abiotic self-replication.

The key to the origins of life is the replication of information. Linear polymers such as nucleic acids that both carry information and can be replicated are currently what we consider to be the basis of living systems. However, these two properties are not necessarily coupled. The ability to mutate in a discrete or quantized way, without frequent reversion, may be an additional requirement for Darwinian evolution, in which case the notion that Darwinian evolution defines life may be less of a tautology than previously thought. In this Account, we examine a variety of in vitro systems of increasing complexity, from simple chemical replicators up to complex systems based on in vitro transcription and translation. Comparing and contrasting these systems provides an interesting window onto the molecular origins of life. For nucleic acids, the story likely begins with simple chemical replication, perhaps of the form A + B → T, in which T serves as a template for the joining of A and B. Molecular variants capable of faster replication would come to dominate a population, and the development of cycles in which templates could foster one another's replication would have led to increasingly complex replicators and from thence to the initial genomes. The initial genomes may have been propagated by RNA replicases, ribozymes capable of joining oligonucleotides and eventually polymerizing mononucleotide substrates. As ribozymes were added to the genome to fill gaps in the chemistry necessary for replication, the backbone of a putative RNA world would have emerged. It is likely that such replicators would have been plagued by molecular parasites, which would have been passively replicated by the RNA world machinery without contributing to it. These molecular parasites would have been a major driver for the development of compartmentalization/cellularization, as more robust compartments could have outcompeted parasite-ridden compartments. The eventual outsourcing of metabolic functions (including the replication of nucleic acids) to more competent protein enzymes would complete the journey from an abiotic world to the molecular biology we see today.

Genetically modifying skin microbe to produce violacein and augmenting microbiome did not defend Panamanian golden frogs from disease.

We designed two probiotic treatments to control chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) on infected Panamanian golden frogs (Atelopus zeteki), a species that is thought to be extinct in the wild due to Bd. The first approach disrupted the existing skin microbe community with antibiotics then exposed the frogs to a core golden frog skin microbe (Diaphorobacter sp.) that we genetically modified to produce high titers of violacein, a known antifungal compound. One day following probiotic treatment, the engineered Diaphorobacter and the violacein-producing pathway could be detected on the frogs but the treatment failed to improve frog survival when exposed to Bd. The second approach exposed frogs to the genetically modified bacterium mixed into a consortium with six other known anti-Bd bacteria isolated from captive A. zeteki, with no preliminary antibiotic treatment. The consortium treatment increased the frequency and abundance of three probiotic isolates (Janthinobacterium, Chryseobacterium, and Stenotrophomonas) and these persisted on the skin 4 weeks after probiotic treatment. There was a temporary increase in the frequency and abundance of three other probiotics isolates (Masillia, Serratia, and Pseudomonas) and the engineered Diaphorobacter isolate, but they subsequently disappeared from the skin. This treatment also failed to reduce frog mortality upon exposure.

Hachimoji DNA and RNA: A genetic system with eight building blocks.

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

Predicting Evolution of the Transcription Regulatory Network in a Bacteriophage.

Prediction of evolutionary trajectories has been an elusive goal, requiring a deep knowledge of underlying mechanisms that relate genotype to phenotype plus understanding how phenotype impacts organismal fitness. We tested our ability to predict molecular regulatory evolution in a bacteriophage (T7) whose RNA polymerase (RNAP) was altered to recognize a heterologous promoter differing by three nucleotides from the wild-type promoter. A mutant of wild-type T7 lacking its RNAP gene was passaged on a bacterial strain providing the novel RNAP in trans. Higher fitness rapidly evolved. Predicting the evolutionary trajectory of this adaptation used measured in vitro transcription rates of the novel RNAP on the six promoter sequences capturing all possible one-step pathways between the wild-type and the heterologous promoter sequences. The predictions captured some of the regulatory evolution but failed both in explaining 1) a set of T7 promoters that consistently failed to evolve and 2) some promoter evolution that fell outside the expected one-step pathways. Had a more comprehensive set of transcription assays been undertaken initially, all promoter evolution would have fallen within predicted bounds, but the lack of evolution in some promoters is unresolved. Overall, this study points toward the increasing feasibility of predicting evolution in well-characterized, simple systems.

Genetic alphabet expansion transcription generating functional RNA molecules containing a five-letter alphabet including modified unnatural and natural base nucleotides by thermostable T7 RNA polymerase variants.

Thermostable T7 RNA polymerase variants were explored for genetic alphabet expansion transcription involving the unnatural Ds-Pa pair. One variant exhibited high incorporation efficiencies of functionally modified Pa substrates and enabled the simultaneous incorporation of 2'-fluoro-nucleoside triphosphates of pyrimidines into transcripts, allowing the generation of novel, highly functional RNA molecules.

Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for in Vivo or in Vitro Synthetic Circuitry.

T7 RNA polymerase is the foundation of synthetic biological circuitry both in vivo and in vitro due to its robust and specific control of transcription from its cognate promoter. Here we present the directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter. These newly described pairs can be used to independently control up to six circuits in parallel.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

Library generation by gene shuffling.

This unit describes the process of gene shuffling, also known as sexual PCR. Gene shuffling is a facile method for the generation of sequence libraries containing the information from a family of related genes. Essentially, related genes are fragmented by DNase I digestion and reassembled by primer-less PCR. The resulting chimeric genes can then be screened or selected for a desired function.

Directed evolution of genetic parts and circuits by compartmentalized partnered replication.

Most existing directed evolution methods, both in vivo and in vitro, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function. To overcome these barriers, here we present compartmentalized partnered replication (CPR). In this approach, synthetic circuits are linked to the production of Taq DNA polymerase so that evolved circuits that most efficiently drive Taq DNA polymerase production are enriched by exponential amplification during a subsequent emulsion PCR step. We apply CPR to evolve a T7 RNA polymerase variant that recognizes an orthogonal promoter and to reengineer the tryptophanyl tRNA-synthetase:suppressor tRNA pair from Saccharomyces cerevisiae to efficiently and site-specifically incorporate an unnatural amino acid into proteins. In both cases, the CPR-evolved parts were more orthogonal and/or more active than variants evolved using other methods. CPR should be useful for evolving any genetic part or circuit that can be linked to Taq DNA polymerase expression.

In vitro selection using modified or unnatural nucleotides.

Incorporation of modified nucleotides into in vitro RNA or DNA selections offers many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions.

An in vitro autogene.

Recent technological advances have allowed development of increasingly complex systems for in vitro evolution. Here, we describe an in vitro autogene composed of a self-amplifying T7 RNA polymerase system. Functional autogene templates in cell-free lysate produce T7 RNA polymerase, which amplifies the autogene genetic information through a positive feedback architecture. Compartmentalization of individual templates within a water-in-oil emulsion links genotype and phenotype, allowing evolution.

Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase.

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.