@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{BrechunArndtWoolley2017, author = {Brechun, Katherine E. and Arndt, Katja Maren and Woolley, G. Andrew}, title = {Strategies for the photo-control of endogenous protein activity}, series = {Current opinion in structural biology : review of all advances ; evaluation of key references ; comprehensive listing of papers}, volume = {45}, journal = {Current opinion in structural biology : review of all advances ; evaluation of key references ; comprehensive listing of papers}, publisher = {Elsevier}, address = {London}, issn = {0959-440X}, doi = {10.1016/j.sbi.2016.11.014}, pages = {53 -- 58}, year = {2017}, language = {en} } @misc{BaumannArndtMueller2013, author = {Baumann, Tobias and Arndt, Katja Maren and M{\"u}ller, Kristian M.}, title = {Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {983}, issn = {1866-8372}, doi = {10.25932/publishup-43108}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-431085}, pages = {13}, year = {2013}, abstract = {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.}, language = {en} } @article{FeinerTeschnerTeschneretal.2019, author = {Feiner, Rebecca Christine and Teschner, Julian and Teschner, Kathrin E. and Radukic, Marco T. and Baumann, Tobias and Hagen, Sven and Hannappel, Yvonne and Biere, Niklas and Anselmetti, Dario and Arndt, Katja Maren and M{\"u}ller, Kristian Mark}, title = {rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations}, series = {International journal of molecular sciences}, volume = {20}, journal = {International journal of molecular sciences}, number = {22}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms20225702}, pages = {19}, year = {2019}, abstract = {Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme beta-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with beta-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.}, language = {en} } @article{BrechunZhenJaikaranetal.2019, author = {Brechun, Katherine E. and Zhen, Danlin and Jaikaran, Anna and Borisenko, Vitali and Kumauchi, Masato and Hoff, Wouter D. and Arndt, Katja Maren and Woolley, Andrew G}, title = {Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo}, series = {Biochemistry}, volume = {58}, journal = {Biochemistry}, number = {23}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.9b00279}, pages = {2682 -- 2694}, year = {2019}, abstract = {We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce similar to 100\% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.}, language = {en} } @article{HoffmannHaoShearwinetal.2019, author = {Hoffmann, Stefan A. and Hao, Nan and Shearwin, Keith E. and Arndt, Katja Maren}, title = {Characterizing transcriptional interference between converging genes in bacteria}, series = {ACS synthetic biology}, volume = {8}, journal = {ACS synthetic biology}, number = {3}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.8b00477}, pages = {466 -- 473}, year = {2019}, abstract = {Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.}, language = {en} } @article{StaabWalossekNellessenetal.2010, author = {Staab, Paul R. and Walossek, J{\"o}rg and Nellessen, David and Gr{\"u}nberg, Raik and Arndt, Katja Maren and M{\"u}ller, Kristian M.}, title = {SynBioWave : a real-time communication platform for molecular and synthetic biology}, issn = {1367-4803}, doi = {10.1093/bioinformatics/btq518}, year = {2010}, abstract = {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.}, language = {en} } @article{SpeckRaeuberKuekenshoeneretal.2013, author = {Speck, Janina and R{\"a}uber, Christina and K{\"u}kensh{\"o}ner, Tim and Niem{\"o}ller, Christoph and Mueller, Katelyn J. and Schleberger, Paula and Dondapati, Padmarupa and Hecky, Jochen and Arndt, Katja Maren and M{\"u}ller, Kristian M.}, title = {TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation}, series = {Protein engineering design \& selection}, volume = {26}, journal = {Protein engineering design \& selection}, number = {3}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {1741-0126}, doi = {10.1093/protein/gzs098}, pages = {225 -- 242}, year = {2013}, abstract = {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.}, language = {en} } @book{Arndt2010, author = {Arndt, Katja Maren}, title = {Proteine zur Krebstherapie - Zielen, Steuern, Hemmen : Antrittsvorlesung 2010-12-08}, publisher = {Univ.-Bibl.}, address = {Potsdam}, year = {2010}, abstract = {Biotechnologie, Biologie, Protein Engineering, Therapeutische Peptide, Protein Design, Selektionssysteme / biotechnology, biology, protein enginieering, therapeutic peptides, protein design, selection systems}, language = {de} } @article{BaumannArndtMueller2013, author = {Baumann, Tobias and Arndt, Katja Maren and M{\"u}ller, Kristian M.}, title = {Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V}, series = {BMC biotechnology}, volume = {13}, journal = {BMC biotechnology}, number = {10}, publisher = {BioMed Central}, address = {London}, issn = {1472-6750}, doi = {10.1186/1472-6750-13-81}, pages = {11}, year = {2013}, abstract = {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.}, language = {en} }