A Logically Reversible Double Feynman Gate with Molecular Engineered Bacteria Arranged in an Artificial Neural Network-Type Architecture.

Reversible logic gates are the key components of reversible computing that map inputs and outputs in a certain one-to-one pattern so that the output signals can reveal the pattern of the input signals. One of the main research foci of reversible computing is the implementation of basic reversible gates by various modalities. Though true thermodynamic reversibility cannot be attained within living cells, the high energy efficiency of biological reactions inspires the implementation of reversible computation in living cells. The implementation of synthetic genetic circuits is mostly based on conventional irreversible computing, and the implementation of logical reversibility in living cells is rare. Here, we constructed a 3-input-3-output synthetic genetic reversible double Feynman logic gate with a population of genetically engineered E. coli cells. Instead of following hierarchical electronic design principles, we adapted the concept of artificial neural networks (ANN) and built a single-layer artificial network-type architecture with five different engineered bacteria, named bactoneurons. We used three extracellular chemicals as input signals and the expression of three fluorescence proteins as the output signals. The cellular devices, which combine the input chemical signals linearly and pass them through a nonlinear activation function and represent specific bactoneurons, were built by designing and creating small synthetic genetic networks inside E. coli. The weights of each of the inputs and biases of individual bactoneurons in the bacterial ANN were adjusted by optimizing the synthetic genetic networks. When arranging the five bactoneurons through an ANN-type architecture, the system generated a double Feynman gate function at the population level. To our knowledge, this is the first reversible double Feynman gate realization with living cells. This work may have significance in development of biocomputer technology, reversible computation, ANN wetware, and synthetic biology.

Synthetic Genetic Reversible Feynman Gate in a Single E. coli Cell and Its Application in Bacterial to Mammalian Cell Information Transfer.

Reversible computing is a nonconventional form of computing where the inputs and outputs are mapped in a unique one-to-one fashion. Reversible logic gates in single living cells have not been demonstrated. Here, we constructed a synthetic genetic reversible Feynman gate in single E. coli cells, and the input-output relations were measured in a clonal population. The inputs were extracellular chemicals, isopropyl ß-d-1-thiogalactopyranoside (IPTG), and anhydrotetracycline (aTc), and the outputs were two fluorescence proteins. We developed a simple mathematical model and simulation to capture the essential features of the circuit and experimentally demonstrated that the behavior of the circuit was ultrasensitive and predictive. We showed an application by creating an intercellular Feynman gate, where input information from bacteria was computed and transferred to HeLa cells through shRNAs delivery and the output signals were observed as silencing of native AKT1 and CTNNB1 genes. The introduction of reversible logics in synthetic biology is new, and given that one-to-one input-output mapping, such reversible genetic systems might have applications in sensing, diagnostics, cellular computing, and synthetic biology.

Synthetic biology: at the crossroads of genetic engineering and human therapeutics-a Keystone Symposia report.

Synthetic biology has the potential to transform cell- and gene-based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease-related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell-based therapies.

A single layer artificial neural network type architecture with molecular engineered bacteria for reversible and irreversible computing.

Here, we adapted the basic concept of artificial neural networks (ANNs) and experimentally demonstrate a broadly applicable single layer ANN type architecture with molecular engineered bacteria to perform complex irreversible computing like multiplexing, de-multiplexing, encoding, decoding, majority functions, and reversible computing like Feynman and Fredkin gates. The encoder and majority functions and reversible computing were experimentally implemented within living cells for the first time. We created cellular devices, which worked as artificial neuro-synapses in bacteria, where input chemical signals were linearly combined and processed through a non-linear activation function to produce fluorescent protein outputs. To create such cellular devices, we established a set of rules by correlating truth tables, mathematical equations of ANNs, and cellular device design, which unlike cellular computing, does not require a circuit diagram and the equation directly correlates the design of the cellular device. To our knowledge this is the first adaptation of ANN type architecture with engineered cells. This work may have significance in establishing a new platform for cellular computing, reversible computing and in transforming living cells as ANN-enabled hardware.

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.

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.