Construction of a Rhythm Transfer System That Mimics the Cellular Clock

Creation of an artificial oscillating gene expression system is one of the most challenging issues in synthetic biology. Here, we constructed a simple system to manipulate gene expression patterns to be circadian, reflecting the intrinsic cellular clock, by fusing a core clock protein, BMAL1 or CLOCK, with a zinc finger-type DNA binding domain. Circadian rhythmic gene expression was induced only when the target gene contained zinc finger-binding sequences. To our knowledge, this simple approach is the first to manipulate gene expression patterns into circadian rhythms and would be applicable to various endogenous genes

Construction of a Ca²⁺-Gated Artificial Channel by Fusing Alamethicin with a Calmodulin-Derived Extramembrane Segment

Using native chemical ligation, we constructed a Ca²⁺-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca²⁺, this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca²⁺ significantly lengthened the specific channel open state and increased the mean channel current, while Mg²⁺ produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca²⁺-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein