Biology subjects

Baumschlager, A.

Publications and source records attributed to Baumschlager, A..

4 recordsLinked to original sources

bioRxiv2022

Enhancing the performance of Magnets photosensors through directed evolution

Photosensory protein domains are the basis of optogenetic protein engineering. These domains originate from natural sources where they fulfill specific functions ranging from the protection against photooxidative damage to circadian rhythms. When used in synthetic biology, the features of these photosensory domains can be specifically tailored towards the application of interest, enabling their full exploitation for optogenetic regulation in basic research and applied bioengineering. In this work, we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity. We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants. For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted. We functionally characterize the variants in vivo in bacteria on the single-cell and the population levels. We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells. We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments. Our findings expand the applicability of the widely used Magnets photosensors by enabling a tuning towards the needs of specific optogenetic regulation strategies. More generally, our approach will aid optogenetic approaches by making the adaptation of photosensor properties possible to better suit specific experimental or bioprocess needs.

synthetic biology↗

bioRxiv2021

Implementation of a novel optogenetic tool in mammalian cells based on a split T7 RNA polymerase

Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to orthogonally control gene expression in mammalian cells. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize and optimize a new orthogonal optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7 - mOptoT7). In our study we exploited the T7 polymerases viral origins to tune our systems expression level, reaching up to 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate gene expression burden when compared to other optogenetic constructs. These properties make mOptoT7 a new powerful tool to use when orthogonality and viral-like RNA species are desired in both synthetic biology and basic science applications.

synthetic biology↗

bioRxiv2020

An inducible AraC that responds to blue light instead of arabinose

In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.

microbiology↗

bioRxiv2017

Dynamic blue light-inducible T7 RNA polymerases (Opto-T7RNAPs) for precise spatiotemporal gene expression control

Light has emerged as control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark-state, high expression strength when induced with blue light, or an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we have created a variant, which returns to the inactive dark-state within minutes, once blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. However, minor alterations should be sufficient to allow their use in other species in which the T7 RNAP polymerase and the light-inducible Vivid regulator were shown to be functional, which comprises other bacterial species and eukaryotes such as mammalian cells or yeast. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation, and allow for a more reliable dynamic control of synthetic and natural gene networks.

synthetic biology↗