Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions

Search for dark matter candidates and large extra dimensions in events with a jet and missing transverse momentum with the ATLAS detector

Open Access, Copyright CERN, for the benefit of the ATLAS collaboration. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited

Cell proliferation inhibited by MyoD1 independently of myogenic differentiation

5sinoneCell growth and differentiation are usually mutually exclusive. Transformation of myoblasts by retroviruses containing the myc oncogene inhibits differentiation, preventing cells from withdrawing from the cell cycle. If cell-cycle withdrawal is a prerequisite for myoblast differentiation, it is probably an early event in terminal cell differentiation, but this has not yet been established. MyoD1 regulates myogenesis. It is expressed only in skeletal muscle, but can convert other cells to muscle cells. The MyoD1 protein, a nuclear phosphoprotein in part similar to the myc family of proteins, is a DNA-binding protein binding to the enhancer sequences of the muscle-specific creatine phosphokinase gene. Thus, introduction of MyoD1 into cells provides a simple approach to study the effect of induction of differentiation on cell growth. In cultured NIH 3T3 cells, inhibition of cell proliferation occurs within 18 hours, and expression of myosin starts after 72 hours. Furthermore, injection of MyoD1 into quiescent NIH 3T3 cells inhibit cell proliferation independently of induction of differentiation. Deletion of the myc-like domain in the MyoD1 gene eliminates the inhibition of DNA synthesis, but substitution of the basic domain with the analogous domain from the E12 transcription factor inhibits growth yet fails to induce differentiation. Inhibition of DNA synthesis, therefore, seems to be controlled separately from myogenic differentiation.noneSorrentino, V. ; Pepperkok, R.; Davis, R. L. ; Ansorge, W. ; Philipson, L.Sorrentino, V.; Pepperkok, R.; Davis, R. L.; Ansorge, W.; Philipson, L