@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{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{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{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} } @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{HoffmannWohltatMuelleretal.2017, author = {Hoffmann, Stefan A. and Wohltat, Christian and Mueller, Kristian M. and Arndt, Katja Maren}, title = {A user-friendly, low-cost turbidostat with versatile growth rate estimation based on an extended Kalman filter}, series = {PLoS one}, volume = {12}, journal = {PLoS one}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0181923}, pages = {5944 -- 5952}, year = {2017}, abstract = {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.}, 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{BrechunWoolleyArndt2017, author = {Brechun, Katherine E. and Woolley, Andrew and Arndt, Katja Maren}, title = {A Bacterial Bandpass Assay for Protein-Protein Interactions}, series = {Protein science : a publication of the Protein Society}, volume = {26}, journal = {Protein science : a publication of the Protein Society}, publisher = {Wiley}, address = {Hoboken}, issn = {0961-8368}, pages = {198 -- 198}, year = {2017}, language = {en} } @article{HoffmannKruseArndt2016, author = {Hoffmann, Stefan A. and Kruse, Sabrina M. and Arndt, Katja Maren}, title = {Long-range transcriptional interference in E-coli used to construct a dual positive selection system for genetic switches}, series = {Nucleic acids research}, volume = {44}, journal = {Nucleic acids research}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0305-1048}, doi = {10.1093/nar/gkw125}, pages = {12}, year = {2016}, abstract = {We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong \&\#963;70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.}, language = {en} } @misc{HoffmannWohltatMuelleretal.2017, author = {Hoffmann, Stefan A. and Wohltat, Christian and M{\"u}ller, Kristian M. and Arndt, Katja Maren}, title = {A user-friendly, low-cost turbidostat with versatile growth rate estimation based on an extended Kalman filter}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-403406}, pages = {15}, year = {2017}, abstract = {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.}, language = {en} }