title: Building blocks for protein interaction devices.
abstract: Here, we propose a framework for the design of synthetic protein networks from modular protein-protein or protein-peptide interactions and provide a starter toolkit of protein building blocks. Our proof of concept experiments outline a general work flow for part-based protein systems engineering. We streamlined the iterative BioBrick cloning protocol and assembled 25 synthetic multidomain proteins each from seven standardized DNA fragments. A systematic screen revealed two main factors controlling protein expression in Escherichia coli: obstruction of translation initiation by mRNA secondary structure or toxicity of individual domains. Eventually, 13 proteins were purified for further characterization. Starting from well-established biotechnological tools, two general-purpose interaction input and two readout devices were built and characterized in vitro. Constitutive interaction input was achieved with a pair of synthetic leucine zippers. The second interaction was drug-controlled utilizing the rapamycin-induced binding of FRB(T2098L) to FKBP12. The interaction kinetics of both devices were analyzed by surface plasmon resonance. Readout was based on Förster resonance energy transfer between fluorescent proteins and was quantified for various combinations of input and output devices. Our results demonstrate the feasibility of parts-based protein synthetic biology. Additionally, we identify future challenges and limitations of modular design along with approaches to address them.
authors.affiliation: EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), UPF, Barcelona and Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain. raik.gruenberg@crg.es
authors.forename: Raik
authors.initials: R
authors.lastname: Grünberg
authors.forename: Tony S
authors.initials: TS
authors.lastname: Ferrar
authors.forename: Almer M
authors.initials: AM
authors.lastname: van der Sloot
authors.forename: Marco
authors.initials: M
authors.lastname: Constante
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D016350:Leucine Zippers; D015202:Protein Engineering / Q000379:methods*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D004247:DNA; D022061:Tacrolimus Binding Protein 1A