Tuning protein expression through promoter and ribosomal binding site sequences ChE130 – April 1, 2015 Induction of tac and lac promoters Expression of human growth hormone tac IPTG lac Design of Synthetic Ribosome Binding Sites Expression of red fluorescent protein Note thousand-fold variation in level of expression H. M. Salis, E. A. Mirsky and C. A. Voigt, Nature Biotechnol. 27, 946 (2009) DNA • RNA Polymerase Promoter strength RNA • Ribosome Protein RBS strength 5 Thermodynamics of Promoter strength Consider binding energies 2 Possible States RNA polymerase genome promoter unbound bound Transcription ∝ PBound Kinney et al, PNAS (2010) Brewster et al. PLoS Comp Biol (2012) 6 DNA • RNA Polymerase Promoter strength RNA • Ribosome Protein RBS strength 7 Thermodynamics of Ribosomal Binding Site Assembly of 30S complex Formation of 70S Complex and Translation Rate Determining Step ΔGmRNA Salis et al. Nature Biotech (2009) ΔGmRNA:ribosome complex Translation ∝ e-βΔG 8 Approaches to Biomolecular Engineering • Model-based (or rational) design Set Objective Model Construct Design Characterize Design Meet Objective? • Combinatorial library construction Set Objective Construct Library Sort & Characterize Library Meet Objective? Model? 9 Construction via DNA technology Amp-selective marker Promoter pZE-Plac_YPet RBS 1. Design primers for specific sequences or to amplify regions to be investigated 2. Construct final DNA plasmid vector to express YPet using bacterial cells (E. coli) Gene encoding for YPet ColE1 ORI E. coli 10 Fluorescent Protein: YPet Fluorescence ∝ YPet Protein Expression 1. Design primers for specific sequences or to amplify regions to be investigated 2. Construct final DNA plasmid vector to express YPet using bacterial cells (E. coli) 3. Characterize the modifications made to either the promoter or RBS using fluorescence PDB: 1GFL Shaner et al. J Cell Sci (2007) 11 Characterization via Fluorescence Model-Based Design Library Construction • 96-well plate reader assay • Fluorescence-activated cell sorter to scan populations Laser Detector Detector Non-fluorescent cell population # of cells – Measure fluorescence for each designed sequence Fluorescent cell population Fluorescence 12 Questions? Ideas? • Promoter – In and to what dynamic range can promoter strength be tuned? – How accurate are the predictions? In which regions are they most accurate? – How can spacing between the -10 and -35 boxes be included in the model? Could the entire promoter sequence be investigated? How many mutations can be made to find interesting results? – Could you design synthetic promoters? Would they be consistent with the model? Are known promoters consistent with the model? • RBS – How important is the location of the RBS for accurate predictions? – How much flexibility is there in designing RBS? Does it highly favor consensus sequences? – Are there other RBS sequences yet to be discovered in E. coli? 13