Genome sequencing2001 Draft of thehuman genome 1000 genomes project initiated in 2008 60x more sequence data than combined previous 25 years
Information stored in the DNAHow much of DNA is on the world:• 4*1035 bp bacterial DNA (5% DNA)• 4*1034 bp plant DNA (1% DNA)• 1*1034 bp animal DNA (1% DNA)• 5*1030 bp viral DNA (50% DA)• 5*1035 bp of DNA on Earth~1 bit/nm3, much higher density than the existing information storage mediaGenome could be rewritten or modified
Chemical synthesis of oligonucleotides annealing ligation Insertion into plasmidIntroductioninto the cell
The assembly of a synthetic M. mycoides genome Assembled in yeast from 1078 overlapping DNA cassettes in three steps: 1. 1080 bp casettes from synthetic oligos for 10 kbp assemblies 2. 10 assemblies were combined into 100 kbp assemblies 3. 11 fragments were combined into the full chromosome D G Gibson et al. Science 2010;329:52-56
The engineering approach of synthetic biology• Application of engineering principles into biological systems• Important engineering principles: – Modularity – Abstraction – Reliability – Predictability – Standardization Adriananoandro et el., Mol.Sys.Biol. 2006
(Synthetic) Biology isnanotechnology that works. Drew Endyhttp://www.arn.org/mm/mm.htm
Synthetic biology as an investigative tool “What I can not create, I do not understand!” How and why are natural systems constructed as they are ? (functional requirements or evolutionary coincidence) Do we understand the function of all components of a device/system?Richard Feynman
Področja uporabe sintezne biologije Synthetic biology contributes to solve problems in • Health/Medicine • Sustainable sources of energy • New materials & bionanomaterials • Information processing • Biosensors • Bioremediation...
Synthetic biology to (re)design products More efficient and sustainable, production: -Drugs to treat malaria -Biofuels -...http://keaslinglab.lbl.gov/application_areas/index.html
Scaffolding of biosynthetic pathway enzymes toenhance the biosynthetic rate Enzyme Enzyme Enzyme Enzyme 3 4 1 2
Improved biosynthesis using DNA scaffold the order of DNA motifs along the program DNA defines the order of bound functional proteins fusion proteins between DNA binding domain and functional domain (e.g. enzymes)Bound enzymes perform sequential reactions
Improved biosynthesis using DNA scaffold 300% yield improvement 15 C resveratrol (mg/l) 10 4CL STS 5 0 2h 6h h 27 4CL::STS fusion 4bp spacer no program 8bp spacer 2bp spacer
Sintezna biologija za obnovljive vire energije Renewable sources of energy CO2 enzymes cellulose sugars CO2 Microbial conversion fuel
Logic-gated nanorobot for drug delivery DNA origami forms a hexagonal barrell split in half Complementary clamps close the barrell Binding sites inside the barrel can be loaded with cargo conjugated to ssDNADouglas et al., Science 2012
Binding to cells depending on thecombination of surface-exposed markers
Nanostructures in nature Transcending the nature Can we bend molecules at will ?
Polypeptide origami• Polypeptides fold into defined tertiary structures• Nature solved the design problem during hundreds of millionsof years of evolution
Design of complex 3D structures Deconstruct the designed structure into smaller rigid independently-folded building elements and Concatenate those elements into the self-assembling polypeptidehttp://bestbridge.net/Eu_en/the-iron-bridge.html
Coiled-coil interactions governing the specificity of coiled-coil formation are well understood coiled-coils are characterized by regular repeating unit of 7 amino acids heptad repeat (a-b-c-d-e-f-g) stability and specificity is provided by residues at defined positions within the heptad repeat
Flexible linker between building elements How can we build complex structures from rigid building blocks ?
Flexible linker between building elements Each edge in the final structure is composed of a dimer
Design of a tetrahedron-forming polypeptide 4 parallel dimers APHP3BCRGCNshAPHP7GCNshP4P5P8BCRP6 2 antiparallel dimersTET12 His6 flexible tetrapeptide linker
Polypeptide production, isolation, assemblyProduction in E.coli Self-assembly SDS-PAGE Dialysis at low polypeptide concentrationProtein purificationAffinity chromatographyHPLC-RP