A genetic toggle switch was studied experimentally and theoretically. We have found an additional kinetic stable state where all the genes express very lowly, which is predicted to be unstable by dynamical systems theory. It can also stably coexist with the other two known stable states, although this coexistence is forbidden in a deterministic dynamical system. We analyze that this nontrivial phenomenon results from the discrete and fluctuate nature in such a small system, by comparing experimental results with modeling results of exact stochastic simulations and differential equations.
α-Zein has received widespread attention owing to its unique solubility, amphipathic, and self-assembly properties, which is because of its high proportion of nonpolar amino acids and unique amino acid sequence. The protein self-assembly is a significant and widely observed phenomenon in many scientific areas such as food and biomedicine, among many industries. In this study, we investigated the self-assembly behavior of α-zein and regulated the morphology and structure of the self-assembled α-zein by varying the experimental parameters like pH, ethanol content, induction time, and α-zein concentration during the self-assembly process in ethanol–water mixtures. The nanospheres and nanofibers were observed under different conditions [nanospheres observed under acidic and strongly alkaline (pH > 10.5) conditions or for ethanol content lower than 65% and higher than 75%; nanofibers observed under weakly alkaline (pH 9.5–10.5) conditions or for 65–75% ethanol concentration for induction duration longer than 24 h]. The morphological and structural analyses of the self-assembled α-zein showed that the self-assembly process was accompanied by the transformation of the morphology and conformation of α-zein. The studies on the self-assembly process and mechanism revealed that α-zein first self-assembled into nanospheres, followed by the nanospheres adhering to shape-beaded fibers and finally fibers, accompanied by a structural transformation from the disordered into ordered state. The nanosphere formation is noted to follow the nucleation-based polymerization, and the nanosphere-mediated mechanisms lead to the formation of nanofibers. Moreover, the hydrophobic interactions, hydrogen bonds, and electrostatic interactions are concluded to drive the α-zein self-assembly. The findings from this study are expected to provide a theoretical basis for expanding the commercial applications of α-zein.
The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.
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