TY - JOUR A1 - Klingstrom, Tomas A1 - Soldatova, Larissa A1 - Stevens, Robert A1 - Roos, T. Erik A1 - Swertz, Morris A. A1 - Müller, Kristian M. A1 - Kalas, Matus A1 - Lambrix, Patrick A1 - Taussig, Michael J. A1 - Litton, Jan-Eric A1 - Landegren, Ulf A1 - Bongcam-Rudloff, Erik T1 - Workshop on laboratory protocol standards for the molecular methods database JF - New biotechnology N2 - Management of data to produce scientific knowledge is a key challenge for biological research in the 21st century. Emerging high-throughput technologies allow life science researchers to produce big data at speeds and in amounts that were unthinkable just a few years ago. This places high demands on all aspects of the workflow: from data capture (including the experimental constraints of the experiment), analysis and preservation, to peer-reviewed publication of results. Failure to recognise the issues at each level can lead to serious conflicts and mistakes; research may then be compromised as a result of the publication of non-coherent protocols, or the misinterpretation of published data. In this report, we present the results from a workshop that was organised to create an ontological data-modelling framework for Laboratory Protocol Standards for the Molecular Methods Database (MolMeth). The workshop provided a set of short- and long-term goals for the MolMeth database, the most important being the decision to use the established EXACT description of biomedical ontologies as a starting point. Y1 - 2013 U6 - https://doi.org/10.1016/j.nbt.2012.05.019 SN - 1871-6784 VL - 30 IS - 2 SP - 109 EP - 113 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Jedrusik-Bode, Monika A1 - Studencka, Maja A1 - Smolka, Christian A1 - Baumann, Tobias A1 - Schmidt, Henning A1 - Kampf, Jan A1 - Paap, Franziska A1 - Martin, Sophie A1 - Tazi, Jamal A1 - Müller, Kristian M. A1 - Krüger, Marcus A1 - Braun, Thomas A1 - Bober, Eva T1 - The sirtuin SIRT6 regulates stress granule formation in C. elegans and mammals JF - Journal of cell science N2 - SIRT6 is a NAD(+)-dependent deacetylase that modulates chromatin structure and safeguards genomic stability. Until now, SIRT6 has been assigned to the nucleus and only nuclear targets of SIRT6 are known. Here, we demonstrate that in response to stress, C. elegans SIR-2.4 and its mammalian orthologue SIRT6 localize to cytoplasmic stress granules, interact with various stress granule components and induce their assembly. Loss of SIRT6 or inhibition of its catalytic activity in mouse embryonic fibroblasts impairs stress granule formation and delays disassembly during recovery, whereas deficiency of SIR-2.4 diminishes maintenance of P granules and decreases survival of C. elegans under stress conditions. Our findings uncover a novel, evolutionary conserved function of SIRT6 in the maintenance of stress granules in response to stress. KW - C. elegans KW - G3BP KW - SIRT6 KW - Sirtuins KW - Stress KW - Stress granules Y1 - 2013 U6 - https://doi.org/10.1242/jcs.130708 SN - 0021-9533 SN - 1477-9137 VL - 126 IS - 22 SP - 5166 EP - + PB - Company of Biologists Limited CY - Cambridge ER - TY - JOUR A1 - Speck, Janina A1 - Räuber, Christina A1 - Kükenshöner, Tim A1 - Niemöller, Christoph A1 - Mueller, Katelyn J. A1 - Schleberger, Paula A1 - Dondapati, Padmarupa A1 - Hecky, Jochen A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation JF - Protein engineering design & selection N2 - The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric proteinprotein interactions enable TAT-dependent translocation of the resistance marker TEM -lactamase (L). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and L, (ii) dimeric or oligomeric proteinprotein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the L, respectively and (iii) heterotrimeric proteinprotein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split L fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections. KW - HiT selection KW - NHR2 KW - TAT selection KW - three hybrid KW - two hybrid Y1 - 2013 U6 - https://doi.org/10.1093/protein/gzs098 SN - 1741-0126 VL - 26 IS - 3 SP - 225 EP - 242 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Staab, Paul R. A1 - Walossek, Jörg A1 - Nellessen, David A1 - Grünberg, Raik A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - SynBioWave : a real-time communication platform for molecular and synthetic biology N2 - Synthetic Biology is advanced by many users and relies on the assembly of genetic elements to devices, systems and finally genomes. SynBioWave is a software suite that enables multiple distributed users to analyze and construct genetic parts in real-time collaboration. It builds on Google Wave and provides an extensible robot-robot-user communication framework, a menu driven user interface, biological data handling including DAS and an internal database communication. We demonstrate its use by implementing robots for gene-data retrieval, manipulation and display. The initial development of SynBioWave demonstrates the power of the underlying Google Wave protocol for Synthetic Biology and lays the foundation for continuous and user-friendly extensions. Specialized wave-robots with a manageable set of capabilities will divide and conquer the complex task of creating a genome in silico. Y1 - 2010 UR - http://bioinformatics.oxfordjournals.org/ U6 - https://doi.org/10.1093/bioinformatics/btq518 SN - 1367-4803 ER - TY - JOUR A1 - Speck, Janina A1 - Hecky, Jochen A1 - Tam, Heng-Keat A1 - Arndt, Katja Maren A1 - Einsle, Oliver A1 - Müller, Kristian M. T1 - Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution JF - Biochemistry N2 - The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation-compensation strategy, which identifies mutations rescuing the activity of a truncated TEM beta-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-N Delta 15C Delta 3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 degrees C in T-m, displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a T-m increased by 15.3 degrees C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation-optimization cycles can exploit stability-trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information. Y1 - 2012 U6 - https://doi.org/10.1021/bi2018738 SN - 0006-2960 VL - 51 IS - 24 SP - 4850 EP - 4867 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Speck, Janina A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Efficient phage display of intracellularly folded proteins mediated by the TAT pathway JF - Protein engineering design & selection N2 - Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function. KW - g3p KW - phagemid display KW - protein design KW - protein engineering KW - selection Y1 - 2011 U6 - https://doi.org/10.1093/protein/gzr001 SN - 1741-0126 VL - 24 IS - 6 SP - 473 EP - 484 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V JF - BMC biotechnology N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. KW - Cohesive ends KW - DNA cleavage KW - Genetic vectors KW - Modified primers KW - Molecular methods KW - Polymerase chain reaction KW - Recombinant Escherichia coli KW - Restriction enzymes Y1 - 2013 U6 - https://doi.org/10.1186/1472-6750-13-81 SN - 1472-6750 VL - 13 IS - 10 PB - BioMed Central CY - London ER - TY - JOUR A1 - Hoffmann, Stefan A. A1 - Wohltat, Christian A1 - Müller, Kristian M. A1 - Arndt, Katja Maren T1 - A user-friendly, low-cost turbidostat with versatile growth rate estimation based on an extended Kalman filter JF - PLoS one N2 - For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness. Y1 - 2017 U6 - https://doi.org/10.1371/JOURNAL.PONE.0181923 SN - 1932-6203 VL - 12 IS - 7 SP - 1 EP - 15 PB - PLoS CY - Lawrence, Kan. ER -