TY - JOUR A1 - Hoffmann, Stefan A. A1 - Hao, Nan A1 - Shearwin, Keith E. A1 - Arndt, Katja Maren T1 - Characterizing transcriptional interference between converging genes in bacteria JF - ACS synthetic biology N2 - 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. KW - gene regulation KW - antisense transcription KW - transcriptional interference KW - mathematical modeling KW - Escherichia coli Y1 - 2019 U6 - https://doi.org/10.1021/acssynbio.8b00477 SN - 2161-5063 VL - 8 IS - 3 SP - 466 EP - 473 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Hoffmann, Stefan A. A1 - Wohltat, Christian A1 - Mueller, 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 SP - 5944 EP - 5952 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Hoffmann, Stefan A. A1 - Kruse, Sabrina M. A1 - Arndt, Katja Maren T1 - Long-range transcriptional interference in E-coli used to construct a dual positive selection system for genetic switches JF - Nucleic acids research N2 - 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 σ70 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. Y1 - 2016 U6 - https://doi.org/10.1093/nar/gkw125 SN - 0305-1048 SN - 1362-4962 VL - 44 PB - Oxford Univ. Press CY - Oxford ER - TY - GEN 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 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 390 Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-403406 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 - TY - JOUR A1 - Mazumder, Mostafizur A1 - Brechun, Katherine E. A1 - Kim, Yongjoo B. A1 - Hoffmann, Stefan A. A1 - Chen, Yih Yang A1 - Keiski, Carrie-Lynn A1 - Arndt, Katja Maren A1 - McMillen, David R. A1 - Woolley, G. Andrew T1 - An Escherichia coli system for evolving improved light-controlled DNA-binding proteins JF - Protein engineering design & selection N2 - Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library. KW - directed evolution KW - fluorescent reporter KW - optogenetics Y1 - 2015 U6 - https://doi.org/10.1093/protein/gzv033 SN - 1741-0126 SN - 1741-0134 VL - 28 IS - 9 SP - 293 EP - 302 PB - Oxford Univ. Press CY - Oxford ER -