Team Heidelberg Philosopher s Stone

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3 Team Heidelberg Philosopher s Stone

4 Delftia acidovorans Delftibactin. A Special Peptide to Recover Gold from Electronic Waste Toxicity of gold ions Au 2+ Au 2+ Au 2+ Secretion of delftibactin Precipitation of solid gold Au 2+ Au 2+

5 Gold. Bacteria Can Recover It from Electronic Waste. Electronic waste Elementary gold Precipitate Neutralize Disolve Delftia acidovorans

6 Delftibactin. Contains Cannot Is a Peptide. Be Non-Proteinogenic Produced by Amino the Amino Acids. Ribosome. Acids. Orn

7 NRPSs. Enzymes for the Synthesis of Peptides Beyond the Ribosome. Non-Ribosomal Peptide Synthetases Ribosomal Synthesis: 22 Amino Acids > 500 Amino Acids

8 Tyrocidine Second Messengers Delftibactin Siderophores Short Peptides. A Myriad of Functions. Recycling Agents Neuromodulators Pigments Immunosuppressants Indigoidine Biosurfactants Antibiotics Sweetener Cytostatics Decontamination Toxins Metal Chelators

9 NRPSs. A Valid Alternative to Chemical Peptide Synthesis. Peptide Speciality Laboratories Chemical Peptide Synthesis Challenging Expensive, especially in large scale Environmental pollution due to usage of strong acids and bases Establish System for Biological Production of NRPs

10 NRPSs. A Beautiful Example of Modularity. Gene Cluster Ribosomal Translation Synthetase Modules Short Peptide = 125 Million possibilities

11 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

12 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

13 Tyrocidine. A Model System to Study the Modularity of NRPSs. Tyrocidine synthetase modules Monomers Tyrocidine Brevibacillus parabrevis

14 Engineered Synthetases. Paving the Way to Novel Synthetic Peptides. Tyrocidine synthetase modules Shuffling Orn Novel synthetase Synthesis Orn Synthetic tripeptide

15 Ornithine fold change Synthetic Peptides. They Can Be Detected by Mass-Spectrometry. Lysis Orn Orn Orn Orn fold increase Control Tripeptide

16 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

17 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

18 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

19 Tag Properties. Universal Easy Detection Tag Quantitative Read-out Small Easy Handling Inert

20 Blue Pigment Tag. Detection of Short Peptides Made Easy. Indigoidine Synthetase Photorhabdus luminescens IndC L-Glutamine Indigoidine Labeled peptide

21 Our Tag Is Functional! Efficient Labeling of Synthetic Peptides. val IndC Val - Ind asn IndC orn val IndC phe orn leu IndC

22 Indigoidine. It s like GFP for Short Peptides! Green Fluorescent Protein. In vivo labeling of proteins Indigoidine. In vivo labeling of short peptides

23 Cloning Standard. High Throughput Creation of a Library of Tagged Short Peptides. Modules IndC Tag other natural T Domains RFC 99 Synthetic T Domains Library HiCT: of 58 engineered High Throughput Synthetases Circular Polymerase Extension Cloning and Transformation Domains Adenylation Thiolation Thioesterase Specificity & Activation Binding Termination

24 Synthetic T Domains. Better than Nature! Determination of crystal structure of IndC variants by igem Team Marburg

25 Mathematical Modeling. Reveals Key Parameters for Synthetic Peptide Production Bacterial Growth Bacterium Glutamine Indigoidine Production cglutamine Bac max b k dim k syn k degi Growth capacity Growth rate Dimerization rate Synthesis rate Degradation rate Indigoidine Practically and structurally fully identifiable d Bac = Bac Parameter ( Bac] variability Bac representing max β diverse experimental data sets dtbac Bac max max d[gln] b = Bac Gln k dt k degi syn k d[cgln] dim k = k dt cglu 2 syn dim + Bac Gln k syn d Ind = cgln 2 k dt [Ind] k dim degi

26 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

27 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

28 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

29 NRPSDesigner. Custom Peptide Design Made Accessible to Anyone.

30 NRPSDesigner. Custom Peptide Design Made Accessible to Anyone.

31 Domain Boundaries. Experimentallyderived Rules Included in the Software. Tool 2 Tool 1 Tool 2 Tool 1 A T TE

32 NRPSDesigner. A New, Comprehensive Database. 225 NRPS Domains 51 NRP Monomers DNA Coding Sequences

33 NRPSDesigner. Interface to igem. Design New Short Peptides Interact with the Registry Add new Pathways to the Database

34 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

35 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

36 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

37 Gold Recycling. Industries Use Chemicals.

38 Feasibility Study. Scaling Short Peptides Synthesis to Industrial Levels is Possible. Constraint-based modeling (FBA) Simulation of fed-batch bioreactor Comparable price to established methods Much more environment-friendly Biological Recycling of Gold is Feasible!

39 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

40 Our Goal. Revolutionize Peptide Synthesis Using NRPSs. Engineering NRPS Peptide Detection Automated Design Industrial Upscaling

41 From Small to Large Scale.Industrial Production Pipeline of Short Peptides. Library Easy Upscaling RFC 100 NRPS Design Primer + Functional Assay TLC validation Validate RFC 57& 99 Paralellized cloning

42 ? On the way to a Future Public Discussion. Shaping the Future of Synthetic Biology Together.

43 Our Achievements Open up NRPS for Synthetic Biology

44 Our Achievements NRPS Demonstration of NRPS modularity on different levels Universal tag enabling NRP labeling in vivo: BBa_K BBa NRPS toolbox comprised of 20 well-characterized parts Novel approach for engineering truly synthetic NRPS domains and modules RFC 99 New Standard for design and production of custom peptides and peptide libraries: RFC 100

45 Thank You. igem, Our Team and Supporters.