Processing two environmental chemical signals with a synthetic genetic IMPLY gate, a 2-input-2-output integrated logic circuit, and a process pipeline to optimize its systems chemistry in Escherichia coli.

Synthetic genetic devices can perform molecular computation in living bacteria, which may sense more than one environmental chemical signal, perform complex signal processing in a human-designed way, and respond in a logical manner. IMPLY is one of the four fundamental logic functions and unlike others, it is an "IF-THEN" constraint-based logic. By adopting physical hierarchy of electronics in the realm of in-cell systems chemistry, a full-spectrum transcriptional cascaded synthetic genetic IMPLY gate, which senses and integrates two environmental chemical signals, is designed, fabricated, and optimized in a single Escherichia coli cell. This IMPLY gate is successfully integrated into a 2-input-2-output integrated logic circuit and showed higher signal-decoding efficiency. Further, we showed simple application of those devices by integrating them with an inherent cellular process, where we controlled the cell morphology and color in a logical manner. To fabricate and optimize the genetic devices, a new process pipeline named NETWORK Brick is developed. This pipeline allows fast parallel kinetic optimization and reduction in the unwanted kinetic influence of one DNA module over another. A mathematical model is developed and it shows that response of the genetic devices are digital-like and are mathematically predictable. This single-cell IMPLY gate provides the fundamental constraint-based logic and completes the in-cell molecular logic processing toolbox. The work has significance in the smart biosensor, artificial in-cell molecular computation, synthetic biology, and microbiorobotics.

Design, Fabrication, and Device Chemistry of a 3-Input-3-Output Synthetic Genetic Combinatorial Logic Circuit with a 3-Input AND Gate in a Single Bacterial Cell.

Advancement of in-cell molecular computation requires multi-input-multi-output genetic logic devices. However, increased physical size, a higher number of molecular interactions, cross-talk, and complex systems level device chemistry limited the realization of such multi-input-multi-output devices in a single bacterial cell. Here, by adapting a circuit minimization and conjugated promoter engineering approach, we created the first 3-input-3-output logic function in a single bacterial cell. The circuit integrated three extracellular chemical signals as inputs and produced three different fluorescent proteins as outputs following the truth table of the circuit. First, we created a noncascaded 1-gate-3-input synthetic genetic AND gate in bacteria. We showed that the 3-input AND gate was digital in nature and mathematically predictable, two important characteristics, which were not reported for previous 3-input AND gates in bacteria. Our design consists of a 128 bp DNA scaffold, which conjugated various protein-binding sites in a single piece of DNA and worked as a hybrid promoter. The scaffold was a few times smaller than the similar 3-input synthetic genetic AND gate promoter reported. Integrating this AND gate with a new 2-input-2-output integrated circuit, which was also digital-like and predictive, we created a 3-input-3-output combinatorial logic circuit. This work demonstrated the integration of a 3-input AND gate in a larger circuit and a 3-input-3-output synthetic genetic circuit, both for the first time. The work has significance in molecular computation, biorobotics, DNA nanotechnology, and synthetic biology.

Dynamic Adaptation in Extant Porins Facilitates Antibiotic Tolerance in Energetic Escherichia coli.

Bacteria can tolerate antibiotics despite lacking the genetic components for resistance. The prevailing notion is that tolerance results from depleted cellular energy or cell dormancy. In contrast to this view, many cells in the tolerant population of Escherichia coli can exhibit motility - a phenomenon that requires cellular energy, specifically, the proton-motive force (PMF). As these motile-tolerant cells are challenging to isolate from the heterogeneous tolerant population, their survival mechanism is unknown. Here, we discovered that motile bacteria segregate themselves from the tolerant population under micro-confinement, owing to their unique ability to penetrate micron-sized channels. Single-cell measurements on the motile-tolerant population showed that the cells retained a high PMF, but they did not survive through active efflux alone. By utilizing growth assays, single-cell fluorescence studies, and chemotaxis assays, we showed that the cells survived by dynamically inhibiting the function of existing porins in the outer membrane. A drug transport model for porin-mediated intake and efflux pump-mediated expulsion suggested that energetic tolerant cells withstand antibiotics by constricting their porins. The novel porin adaptation we have uncovered is independent of gene expression changes and may involve electrostatic modifications within individual porins to prevent extracellular ligand entry.

A microgravity responsive synthetic genetic device in Escherichia coli.

Bioengineering solutions to human space travel must consider microgravity as an important component. Thus, one of the fundamental challenges of space bioengineering is to create cellular microgravity responsive device, which integrate microgravity as a signal within biochemical and cellular processes. Here, we designed, fabricated and characterized the first biochemical and cellular microgravity responsive device using an engineered genetic circuit in E.coli, which responded to microgravity by changing the expression of a target enhanced green fluorescent gene (EGFP). Our device design was based on the deregulation of HfQ protein in E.coli in microgravity, which was translated through HfQ mediated silencing of EGFP by anti-EGFP synthetic small regulatory RNAs. This resulted a reduced silencing (~28 times) of the EGFP in microgravity. We demonstrated that the basic design of the device is universal in nature for E.coli, by creating multiple successful devices, where target genes (EGFP, TdTomato, and FtsZ) and the promoters (inducible and constitutive) were altered. Further, we applied this device to control the cell division process by microgravity. Here we targeted the cell division regulator FtsZ, which resulted an elongated cell shape in normal gravity and this deformed cell shape got rescued to normal one by applying microgravity. The work showed for the first time, a way to integrate microgravity as a physical signal within biochemical processes of a living cell in a human designed way and thus, may have significance in space bioengineering and synthetic biology.

A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits.

BACKGROUND: Frame-shifted genes results in non-functional peptides. Because of this complete loss of function, frame-shifted genes have never been used in constructing synthetic gene circuits. RESULTS: Here we report that the function of gene circuits is rescued by a frame-shifted gene, which functions by translating from a non-natural start codon. We report a single nucleotide deletion mutation that developed in the λ-repressor cI within a synthetic genetic NOT gate in Escherichia coli during growth and through this mutation, a non-functional synthetic gene circuit became functional. This mutation resulted in a frame-shifted cI, which showed effective functionality among genetic NOT-gates in Escherichia coli with high regulatory ranges (> 300) and Hill coefficient (> 6.5). The cI worked over a large range of relative copy numbers between the frame-shifted gene and its target promoter. These properties make this frame-shifted gene an excellent candidate for building synthetic gene circuits. We hypothesized a new operating mechanism and showed evidence that frame-shifted cI was translated from non-natural start codon. We have engineered and tested a series of NOT gates made from a library of cI genes, each of which starts from a different codon within the first several amino acids of the frame-shifted cI. It is found that one form with start codon ACA, starting from the 3rd codon had similar repression behavior as the whole frame-shifted gene. We demonstrated synthetic genetic NAND and NOR logic-gates with frame-shifted cI. This is the first report of synthetic-gene-circuits made from a frame-shifted gene. CONCLUSIONS: This study inspires a new view on frame-shifted gene and may serve as a novel way of building and optimizing synthetic-gene-circuits. This work may also have significance in the understanding of non-directed evolution of synthetic genetic circuits.

A systems biology pipeline identifies new immune and disease related molecular signatures and networks in human cells during microgravity exposure.

Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions.