GRK 2062:  Molecular Principlas of Synthetic Biology

• Subject Area: Basic Research in Biology and Medicine
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 252961272

Final Report Abstract

Biological systems are constructed from elementary building blocks, and only their manifold and dynamic interplay provides the basis for life. To really understand this complexity, scientists need new approaches and tools. In this context, synthetic biology plays an important role because it focuses on the (re-)design of biological systems. This discipline is marked by highly creative approaches and requires a high degree of interdisciplinarity. The RTG2062 was built on internationally recognized working groups in biology, chemistry, and physics that were both experimentally and theoretically oriented and throve on interdisciplinary collaboration. This combination of factors allowed RTG2062 to offer a unique platform for emulating biological systems, creating functional synthetic modules, and training junior researchers in this new way of thinking and approaching research. RTG2062 research gained in visibility, as demonstrated by the successful International “Synthetic Biology” conference series “Synthetic Biology” (Synthetic Biology III in 2018), unique for the wide range of topics it covered. Still, RTG2062 was unable for covering all possible research areas in synthetic biology. Therefore, we decided to concentrate our activities in three research areas: generating synthetic cells and switches (area A), rationally designing and synthesizing molecular building blocks (area B), and reconstructing isolated biological parts and modules (area C). While at first glance these projects seem highly diverse, they all shared the same drive toward unravelling nature’s design principles for a deeper understanding of processes like photosynthesis, translational regulation, cell polarity and coat formation in prokaryotes. In addition, we were developing standardized biological parts, such as designer proteins and peptides, for potential application in medicine and biotechnology. Collaboration between disciplines allowed, for example, synthesis of nucleotides/genes, amino acids, and glyco-peptides by the chemistry groups. These compounds were required in projects addressing biological questions. Similarly, experts in gene expression worked together with plant scientists. It is this interdisciplinary work that has allowed us, on the one hand, to gain new insights into the design principles and mechanisms of nature and, on the other hand, to produce synthetic macromolecular units with new properties. Concurrently, RTG2062 offered junior researchers in the early stages of their careers vital methodological and conceptual training. The success of this unique setting resulted in the successful completion of 19 doctoral theses and 80 publications in high-impact journals. Particularly the numerous joint publications are an important indicator of how effective our bringing together groups from different disciplines has been in achieving synergistic progress in this new field of research.

Publications

• (2015). CRISPR - Cas: From the bacterial adaptive immune system to a versatile genome engineering toolbox. Angew Chem Int Ed Engl 54, 13508-13514
  Kirchner, M., and Schneider, S.
  
• (2016). Chemical communication between bacteria and cell-free gene expression systems within linear chains of emulsion droplets. Integr Biol 8, 564-570
  Schwarz-Schilling, M., Aufinger, L., Muckl, A., and Simmel, F.C.
  
• (2016). One-pot synthesis of functionalized β-fluoroalkylated Mannich-type products from N-aryl N,O-acetals. Synthesis 48, 1167-1176
  Wunder, S., Deutsch, A., Deutsch, C., and Hoffmann-Röder, A.
  
• (2016). Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P (EF-P) triggers generation of the first ArgRha specific antibody. Chem Sci 7, 6995-7001
  Li, X., Krafczyk, R., Macoşek, J., Li, Y.-L., Zou, Y., Simon, B., Pan, X., Wu, Q.-Y., Yan, F, Li, S., Hennig, J., Jung, K., Lassak, J., and Hu, H.-G.
  
• (2016). The Arabidopsis protein CGLD11 is required for chloroplast ATP synthase accumulation. Mol Plant 9, 885-899
  Grahl, S., Reiter, B., Gügel, I.L., Vamvaka, E., Gandini, C., Jahns, P., Soll, J., Leister, D. and Rühle, T.
  
• (2016). The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 in Arabidopsis is required for efficient manganese uptake at the thylakoid membrane. The Plant Cell 28, 892-910
  Schneider, A., Steinberger, I., Herdean, A., Gandini, C., Eisenhut, M., Kurz, S., Morper, A., Hoecker, N., Rühle T., Labs, M., Flügge, U. I., Geimer, S., Schmidt, S. B., Husted, S., Weber, A.P.M., Spetea, C. and Leister D.
  
• (2017). A versatile toolbox for the control of protein levels using Nε-acetyl-L-lysine dependent amber suppression. ACS Synth Biol 6, 1892-1902
  Volkwein, W., Maier, C., Krafczyk, R., Jung, K., and Lassak, J.
  
• (2017). An in vivo highthroughput screening for riboswitch ligands using a reverse reporter gene system. Sci Rep 7, 7732
  Kirchner, M., Schorpp, K., Hadian, K., and Schneider, S.
  
• (2017). Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci Rep 7, 14134
  Radeck, J., Meyer, D., Lautenschläger, N., and Mascher, T.
  
• (2017). Cell-free protein synthesis in micro compartments: building a minimal cell from biobricks. N Biotechnol 39, 199-205
  Jia, H., Heymann, M., Bernhard, F., Schwille, P., and Kai, L.
  
• (2017). CRISPR-Cas9: from a bacterial immune system to genome-edited human cells in clinical trials. Bioengineered 8, 280-286
  Kick, L., Kirchner, M., and Schneider, S.
  
• (2017). Enhancing (crop) plant photosynthesis by introducing novel genetic diversity. Phil Trans R Soc B 372, 20160380
  Dann, M., and Leister, D.
  
• (2017). Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter. Nucleic Acids Res 45, 11033–11042
  Kitsera, N., Allgayer, J., Parsa, E., Geier, N., Rossa, M., Carell, T., and Khobta, A.
  
• (2017). Gene expression control by Bacillus anthracis purine riboswitches. RNA 23, 762-769
  Kirchner, M., and Schneider, S.
  
• (2017). N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis. Nat Struct Mol Biol 24, 870–878
  Edupuganti, R.R., Geiger, S., Lindeboom, R.G.H., Shi, H., Hsu, P.J., Lu, Z., Wang, S.-Y., Baltissen, M.P.A., Jansen, P.W.T.C., Rossa, M., Müller, M., Stunnenberg, H. G., He, C., Carell, T., and Vermeulen, M.
  
• (2017). Rational design of an anticalin-type sugar-binding protein using a genetically encoded boronate side chain. ACS Synth Biol 6, 2241-2247
  Edwardraja, S., Eichinger, A., Theobald, I., Sommer, C.A., Reichert, A.J., and Skerra, A.
  
• (2017). Structural basis for EarP- mediated arginine glycosylation of translation elongation factor EF-P. mBio 8, e01412-17
  Krafczyk, R., Macošek, J., Jagtap, P.K.A., Gast, D., Wunder, S., Mitra, P., Jha, A.K., Rohr, J., Hoffmann-Röder, A., Jung, K., Hennig, J., and Lassak, J.
  
• (2017). The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis. Sci Rep 7, 15058
  Popp, P.F., Dotzler, M., Radeck, J., Bartels, J., and Mascher, T.
  
• (2017). The transporter SynPAM71 is located in the plasma membrane and thylakoids, and mediates manganese tolerance in Synechocystis PCC6803. New Phytol 215, 256-268
  Gandini, C., Schmidt, S.B., Husted, S., Schneider, A., and Leister, D.
  
• Bacillus anthracis purine riboswitches: functional characterization and screening for novel ligands. 2017
  Kirchner, Marion
  
• Exploring Synechocystis PCC 6803 as a synthetic biology platform to study plant photosynthesis. 2017
  Gandini, Chiara
  
• (2018). Bacterial chromosome organization by collective dynamics of SMC condensins. J Royal Soc Interface 15, 20180495
  Miermans, C.A., and Broedersz, C.P.
  
• (2018). Capping protein-controlled actin polymerization shapes lipid membranes. Nat Commun 9, 1630
  Dürre, K., Keber, F.C., Bleicher, P., Brauns, F., Cyron, C.J., Faix, J., and Bausch, A.R.
  
• (2018). Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach. PLoS One 13, e0198058
  Mückl, A., Schwarz-Schilling, M., Fischer, K., and Simmel, F.C.
  
• (2018). Functional surface-immobilization of genes using multistep strand displacement lithography. J Vis Exp 140, 58634
  Pardatscher, G., Schwarz-Schilling, M., Sagredo, S., and Simmel, F.C.
  
• (2018). Gene expression on DNA biochips patterned with strand-displacement lithography. Angew Chem Int Ed Engl 57, 4783-4786
  Pardatscher, G., Schwarz-Schilling, M., Daube, S.S., Bar-Ziv, R.H., and Simmel, F.C.
  
• (2018). In vitro reconstitution of self-organizing protein patterns on supported lipid bilayers. J Vis Exp 137, e58139
  Ramm B., Glock P., and Schwille P.
  
• (2018). Konformationsbewegungen von aktiven Membrantransportern. BIOspektrum 24, 495-497
  Mächtel, R., Gebhardt, C., and Cordes, T.
  
• (2018). Light-induced printing of protein structures on membranes in vitro. Nano Lett 18, 7133-7140
  Jia, H., Kai, L., Heymann, M., García-Soriano, D.A., Härtel, T., and Schwille, P.
  
• (2018). Non-canonical bases in the genome: the regulatory information layer in DNA. Angew Chem Int Ed Engl 57, 4296–4312
  Carell, T., Kurz, M.Q., Müller, M., Rossa, M., and Spada, F.
  
• (2018). Optical control of a biological reaction-diffusion system. Angew Chem Int Ed Engl 57, 2362–2366
  Glock, P., Broichhagen, J., Kretschmer, S., Blumhardt, P., Mücksch, J., Trauner, D., and Schwille, P.
  
• (2018). Optimized assembly of a multifunctional RNA-protein nanostructure in a cellfree gene expression system. Nano Lett 18, 2650-2657
  Schwarz-Schilling, M., Dupin, A., Chizzolini, F., Krishnan, S., Mansy, S.S., and Simmel, F.C.
  
• (2018). Self-organisation principles of intracellular pattern formation. Phil Trans R Soc B 373, 20170107
  Halatek, J., Brauns, F., and Frey, E.
  
• (2018). Sporobeads: The utilization of the Bacillus subtilis endospore crust as a protein display platform. ACS Synth Biol 7, 452-461
  Bartels, J., López Castellanos, S., Radeck, J., and Mascher, T.
  
• (2018). Stationary patterns in a two-protein reaction-diffusion system. ACS Synth Biol 8, 148-157
  Glock, P., Ramm, B., Heermann, T., Kretschmer, S., Schweizer, J., Mücksch, J., Alagöz, G., and Schwille, P.
  
• (2018). Structural basis for the specific cotranslational incorporation of p-boronophenylalanine into biosynthetic proteins. Biochemistry 57, 2597-2600
  Schiefner, A., Nästle, L., Landgraf, M., Reichert, A. J., and Skerra, A.
  
• (2018). Switching protein patterns on membranes. Curr Opin Colloid Interface Sci 38, 100-107
  Glock, P., and Schwille, P.
  
• (2018). The MinDE system is a generic spatial cue for membrane protein distribution in vitro. Nat Commun 9, 3942
  Ramm, B., Glock, P., Mücksch, J., Blumhardt, P., Heymann, M., and Schwille, P.
  
• Exceptionally sweet - Studies on the bacterial arginine rhamnosyltransferase EarP. 2018
  Krafczyk, Ralph
  
• Expression of the plant Photosystem II core proteins in the cyanobacterium Synechocystis sp. PCC6803 and characterization of the DEAD-box RNA helicase RH50 of A. thaliana. 2018
  Paieri, Francesca
  
• (2019). A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria. EMBO J 38, e101650
  Peschek, N., Hoyos, M., Herzog, R., Forstner, K.U., and Papenfort, K.
  
• (2019). An integrated transport mechanism of the maltose ABC importer. Res Microbiol 170, 321-337
  Mächtel, R., Narducci, A., Griffith, D.A., Cordes, T., and Orelle, C.
  
• (2019). Bottom-up synthetic biology: reconstitution in space and time. Curr Opin Biotechnol 60, 179-187
  Jia, H., and Schwille, P.
  
• (2019). Design of biochemical pattern forming systems from minimal motifs. eLife 8, e48646
  Glock, P., Brauns, F., Halatek, J., Frey, E., and Schwille, P.
  
• (2019). Design of sealable custom-shaped cell mimicries based on selfassembled monolayers on CYTOP polymer. ACS Appl Mater Interfaces 11, 21372-21380
  Eto, H., Soga, N., Franquelim, H.G., Glock, P., Khmelinskaia, A., Kai, L., Heymann, M., Noji, H., and Schwille, P.
  
• (2019). Evidence that cyanobacterial Sll1217 functions analogously to PGRL1 in enhancing PGR5-dependent cyclic electron flow. Nat Commun 10, 5299
  Dann, M., and Leister, D.
  
• (2019). Ribosomal peptides and small proteins on the rise. ChemBioChem. 20, 1479-86
  Scheidler, C., Kick, L., and Schneider, S.
  
• (2019). Switching the post-translational modification of translation elongation factor EF-P. Front Microbiol 10, 1148
  Volkwein, W., Krafczyk, R., Kumar, P.A.J., Parr, M., Mankina, E., Macošek, J., Guo, Z., Fürst, M.J.L.J., Pfab, M., Frishman, D., Hennig, J., Jung, K., and Lassak, J.
  
• (2019). Temperature-sensitive protein expression in protocells. Chem Commun 55, 6421-6424
  Jia, H., Heymann, M., Härtel, T., Kai, L., and Schwille, P.
  
• (2019). Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides. Science 366, 76-82
  Becker, S., Feldmann, J., Wiedemann, S., Okamura, H., Schneider, C., Iwan, K., Crisp, A., Rossa, M., Amatov, T., and Carell, T.
  
• Artificial multicellular assemblies - Complex dynamics in spatially organized gene circuits. 2019
  Dupin, Aurore
  
• Cell-free expression systems: a bottom-up approach to synthetic biology. 2019
  Schwarz-Schilling, Matthaeus
  
• Controlling and reshaping biological reaction-diffusion. 2019
  Glock, Philipp
  
• Engineering 4D regulation toolbox to control spatiotemporal cell-free reconstitution. 2019
  Jia, Haiyang
  
• (2020). A lattice kinetic Monte-Carlo method for simulating chromosomal dynamics and other (non-)equilibrium bio-assemblies. Soft Matter 16, 544-556
  Miermans, C.A., and Broedersz, C.P.
  
• (2020). A set of rhamnosylation-specific antibodies enables detection of novel protein glycosylations in bacteria. Org Biomol Chem 18, 6823-6828
  Gast, D., Koller, F., Krafczyk, R., Bauer, L., Wunder, S., Lassak, J., and Hoffmann-Röder, A.
  
• (2020). Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET
  Peter, M.F., Gebhardt, C., Mächtel, R., Glaenzer, J., Thomas, G.H., Cordes, T., and Hagelueken, G.
  
• (2020). Elongation factor P is required for EIIGlc translation in Corynebacterium glutamicum due to an essential polyproline motif. Mol Microbiol 115, 320-331
  Pinheiro, B., Petrov, D.P., Guo, L., Martins, G.B., Bramkamp, M., and Jung, K.
  
• (2020). Flow induced symmetry breaking in a conceptual polarity model. Cells 9, 1524
  Wigbers, M.C., Brauns, F., Leung, C. Y., and Frey, E.
  
• (2020). Gene autoregulation by 3‘ UTR-derived bacterial small RNAs. eLife 9, e58836
  Hoyos, M., Huber, M., Forstner, K.U., and Papenfort, K.
  
• (2020). Genetic code expansion, protein expression and protein functionalisation in Bacillus subtilis. ACS Synthetic Biol. 9, 486-493
  Scheidler, C., Vrabel, M., and Schneider, S.
  
• (2020). Phase-space geometry of mass-conserving reaction–diffusion dynamics. Phys Rev X 10, 041036
  Brauns, F., Halatek, J., and Frey, E.
  
• (2020). Shaping giant membrane vesicles in 3D‐printed protein hydrogel cages. Small 16, 1906259
  Jia, H., Litschel, T., Heymann, M., Eto, H., Franquelim, H.G., and Schwille, P.
  
• (2020). Single cell characterization of a synthetic bacterial clock with a hybrid feedback loop containing dCas9-sgRNA. ACS Synth Biol 9, 3377-3387
  Henningsen, J., Schwarz-Schilling, M., Leibl, A., Gutierrez, J.N., Sagredo, S., and Simmel, F.C.
  
• (2020). Structure and function of a novel elongation factor P subfamily in Actinobacteria. Cell Rep 30, 4332-4342.e5
  Pinheiro, B., Scheidler, C.M., Kielkowski, P., Schmid, M., Forné, I., Ye, S., Reiling, N., Takano, E., Imhof, A., Sieber, S. A., Schneider, S., and Jung, K.
  
• (2020). Synthetic post-translational modifications of elongation factor P using the ligase EpmA. FEBS J 288, 663-677
  Pfab, M., Kielkowski, P., Krafczyk, R., Volkwein, W., Sieber, S.A., Lassak, J., and Jung, K.
  
• Computational physics of bacterial chromosome organization by condensin. 2020
  Miermans, Christiaan Adrianus
  
• Establishing a genetic code expansion system in Bacillus subtilis. 2020
  Scheidler, Christopher
  
• Mechanisms of photosynthetic high-light tolerance – Synechocystis sp. PCC6803 as an experimental platform. 2020
  Dann, Marcel
  
• On the role of stochasticity in biochemical systems far from thermal equilibrium: How fluctuations affect phenomenology. 2020
  Wilke, Patrick
  
• Principles of RNA-based gene expression control in Vibrio cholerae. 2020
  Hoyos, Mona
  
• Strategies for synthetic activation of translation elongation factor P. 2020
  Volkwein, Wolfram
  
• The translation elongation factor P in Actinobacteria. 2020
  Pinheiro Damasceno Florentino, Bruno
  
• (2021) Probing biomolecular interactions by a pattern-forming peptide–conjugate sensor. Bioconjugate Chem 32,1, 172-181
  Heermann, T., Franquelim, H.G., Glock, P., Harrington, L., and Schwille, P.
  
• (2021). A hierarchy of protein patterns robustly decodes cell shape information. Nat Phys 17, 578-584
  Wigbers, M. C., Tan, T. H., Brauns, F., Liu, J., Swartz, S. Z., Frey, E., and Fakhri, N.
  
• (2021). Bulksurface coupling identifies the mechanistic connection between Min-protein patterns in vivo and in vitro. Nat Commun 12, 1-11
  Brauns, F., Pawlik, G., Halatek, J., Kerssemakers, J., Frey, E., and Dekker, C.
  
• (2021). Fine-tuning protein self-organization by orthogonal chemo-optogenetic tools. Angew Chem Int Ed Engl 60, 4501-4506
  Sun, H., Jia, H., Ramirez-Diaz, D.A., Budisa, N., and Schwille, P.
  
• (2021). Transcriptional regulation of the Nε-fructoselysine metabolism in Escherichia coli by global and substrate-specific cues. Mol Microbiol 115, 175-190
  Graf von Armansperg, B., Koller, F., Gericke, N., Hellwig, M., Jagtap, P. K. A., Heermann, R., Hennig, J., Henle, T., and Lassak, J.
  
• (2021). Two RmlC paralogs catalyse dTDP-4-keto-6-deoxy-D- glucose epimerization in Pseudomonas putida KT2440. Sci Rep 11, 11991
  Koller, F., and Lassak, J.
  
• (2021). Wavelength selection by interrupted coarsening in reaction-diffusion systems. Phys Rev Lett. 126, 104101
  Brauns, F., Weyer, H., Halatek, J., Yoon, J., and Frey, E.
  
• Function and distribution of protein monoglycosylations in bacteria. 2021
  Koller, Franziska
  
• Principles and theory of protein-based pattern-formation. 2021
  Brauns, Fridtjof
  
• Studien zu Ursprung, Funktion und Sequenzierung von RNA- und DNA-Nukleosiden. 2021
  Rossa, Martin
  
• Synthesis of fluorinated S. pneumoniae serotype 8 glycotope mimetics for synthetic vaccine candidates and development of rhamnosylation-specific antibodies for detection of novel protein glycosylation in bacteria. 2021
  Gast, Daniel