@article{UllnerAresMorellietal.2012, author = {Ullner, E. and Ares, S. and Morelli, L. G. and Oates, A. C. and J{\"u}licher, F. and Nicola, E. and Heussen, R. and Whitmore, D. and Blyuss, K. and Fryett, M. and Zakharova, A. and Koseska, A. and Nene, N. R. and Zaikin, Alexei}, title = {Noise and oscillations in biological sysems multidisciplinary approach between experimental biology, theoretical modelling and synthetic biology}, series = {International journal of modern physics : B, Condensed matter physics, statistical physics, applied physics}, volume = {26}, journal = {International journal of modern physics : B, Condensed matter physics, statistical physics, applied physics}, number = {25}, publisher = {World Scientific}, address = {Singapore}, issn = {0217-9792}, doi = {10.1142/S0217979212460095}, pages = {12}, year = {2012}, abstract = {Rapid progress of experimental biology has provided a huge flow of quantitative data, which can be analyzed and understood only through the application of advanced techniques recently developed in theoretical sciences. On the other hand, synthetic biology enabled us to engineer biological models with reduced complexity. In this review we discuss that a multidisciplinary approach between this sciences can lead to deeper understanding of the underlying mechanisms behind complex processes in biology. Following the mini symposia "Noise and oscillations in biological systems" on Physcon 2011 we have collected different research examples from theoretical modeling, experimental and synthetic biology.}, language = {en} } @article{GorochowskiAycilarKucukgozeBovenbergetal.2016, author = {Gorochowski, Thomas E. and Aycilar-Kucukgoze, Irem and Bovenberg, Roel A. L. and Roubos, Johannes A. and Ignatova, Zoya}, title = {A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes}, series = {ACS synthetic biology}, volume = {5}, journal = {ACS synthetic biology}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.6b00040}, pages = {710 -- 720}, year = {2016}, abstract = {Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources in executing orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exposure to additional ambient stress. Our results demonstrate that a simple model of resource allocation faithfully captures the redistribution of protein synthesis resources when faced with the burden of synthetic gene expression and environmental stress. The tractable nature of the model makes it a versatile tool for exploring the guiding principles of efficient heterologous expression and the indirect interactions that can arise between synthetic circuits and their host chassis because of competition for shared translational resources.}, language = {en} } @article{MachensBalazadehMuellerRoeberetal.2017, author = {Machens, Fabian and Balazadeh, Salma and M{\"u}ller-R{\"o}ber, Bernd and Messerschmidt, Katrin}, title = {Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae}, series = {Frontiers in Bioengineering and Biotechnology}, volume = {5}, journal = {Frontiers in Bioengineering and Biotechnology}, publisher = {Frontiers}, address = {Lausanne}, issn = {2296-4185}, doi = {10.3389/fbioe.2017.00063}, pages = {1 -- 11}, year = {2017}, abstract = {Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host's endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.}, language = {en} } @article{NaseriBalazadehMachensetal.2017, author = {Naseri, Gita and Balazadeh, Salma and Machens, Fabian and Kamranfar, Iman and Messerschmidt, Katrin and M{\"u}ller-R{\"o}ber, Bernd}, title = {Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae}, series = {ACS synthetic biology}, volume = {6}, journal = {ACS synthetic biology}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.7b00094}, pages = {1742 -- 1756}, year = {2017}, abstract = {Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.}, language = {en} } @article{BrechunArndtWoolley2018, author = {Brechun, Katherine Emily and Arndt, Katja Maren and Woolley, G. Andrew}, title = {Selection of protein-protein interactions of desired affinities with a bandpass circuit}, series = {Journal of molecular biology : JMB}, volume = {431}, journal = {Journal of molecular biology : JMB}, number = {2}, publisher = {Elsevier}, address = {London}, issn = {0022-2836}, doi = {10.1016/j.jmb.2018.11.011}, pages = {391 -- 400}, year = {2018}, abstract = {We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to beta-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for beta-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.e. the circuit therefore acts as a bandpass filter for protein-protein interactions. We show that the circuit can be used to recover protein-protein interactions of desired affinity from a mixed population with a range of affinities. The circuit can also be used to select for inhibitors of protein-protein interactions of defined strength. (C) 2018 Elsevier Ltd. All rights reserved.}, language = {en} } @article{delaCruzMachensMesserschmidtetal.2019, author = {de la Cruz, Jorge Gonzalez and Machens, Fabian and Messerschmidt, Katrin and Bar-Even, Arren}, title = {Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast}, series = {ACS synthetic biology}, volume = {8}, journal = {ACS synthetic biology}, number = {5}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.8b00464}, pages = {911 -- 917}, year = {2019}, abstract = {One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.}, language = {en} }