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Evolution and synthetic biology Eörs Szathmáry Collegium Budapest Eötvös University To find out why life is as it is • Optimal solurion among alternatives • Historical contigency (lack of alternatives at the time of evolution) • Vestigial traits (adaptive only in the past, but now frozen) • Understanding by doing (engineering—the airplane MUST fly) • Compare with synthetic organic chemistry The ways of synthetic biology • DNA technology (modify natural systems with some synthetic components) • To perform something natural with unnatural organic components • At the extreme to create unnatural chemical systems with biological properties • Unnatural assembly of natural modules (repressilator) The extended genetic alphabet Why is aminoA not used? The importance of repeating charge Artificial synthesis of the new base pair dZ:dP • Several polymerase enzymes accept it • Can be used in PCR reactions New base pair in the code for new amino acid • Implementation with isoC:isoG pair • UAG nonsense codon for iodotyrosine • Or the (iso-C)AG codon • Challenge: coupling of non-standard amino acids to nonstandard tRNAs by nonstandard synthetases Fisher’s (1930) question "No practical biologist interested in sexual reproduction would be led to work out the detailed consequences experienced by organisms having three or more sexes; yet what else should he do if he wishes to understand why the sexes are, in fact, always two?" •We must go back to the RNA world •RNA was both enzyme and genetic material •The alphabet was optimal then! •More than 3 billion years ago… Some RNA molecules act as enzymes (ribozymes) today The fitness of ribo-organisms W(N) = A(N) Q(N) (vö. Eigen, 1971) W fitness A reproduction rate Q fidelity of replication N size of the alphabet W(N) = A(N) Q(N) There is somewhere an optimal N Von Kiedrowski’s replicator Peptide replicator networks Towards Systems Chemistry • Analysis of synthesis of coupled autocatalytic chemical systems • Origin of life and chemical technology E - FLUX Fet Open Contract n° FP7-225167 History III: Gánti and fluid automata Pathways of supersystem evolution metabolism MB boundary MT template BT MBT INFRABIOLOGICAL SYSTEMS The stochastic corrector model for compartmentation Szathmáry, E. & Demeter L. (1987) Group selection of early replicators and the origin of life. J. theor Biol. 128, 463-486. Grey, D., Hutson, V. & Szathmáry, E. (1995) A re-examination of the stochastic corrector model. Proc. R. Soc. Lond. B 262, 29-35. E - FLUX Fet Open Contract n° FP7-225167 History II: The stochastic corrector model and Andrew’s letter Theory Experiment In vitro compartmentalization E - FLUX Fet Open Contract n° FP7-225167 Integrated digital microfluidic system for directed evolution Droplet production Droplet reinjection Droplet incubation Co-encapsulation of gene and amplification system Gene amplification Droplet pairing Fluorescence detection Droplet sorting Pair gene containing droplet (small droplet) with in vitro transcription/ribozyme substrate mixture (large droplet) Activity Droplet fusion No activity Detection of ribozyme activity and selection of droplets exceeding desired threshold Droplet incubation Ribozyme expression and activity E - FLUX Fet Open Contract n° FP7-225167 Model system: ribozyme RNase X motif Fluorescence-based assay and real time monitoring + X motif Products Atto488 Quencher Substrate - X motif X motif Cleavage Orthogonal systems Substrate A X motif A Substrate B X motif B Substrate C X motif C E - FLUX Fet Open Contract n° FP7-225167 Qb replicase amplification and ribozyme activity Purification and activity of the Qb replicase Crude extract Qb replicase Activity test Purification Amplified MDV RNA b-subunit EF-Tu EF-Ts Qb-X motif A X motif embedded in MDV retains its activity Substrate A Product A Towards... • Division of labour between genes and enzymes • Origin of chromosomes by spontaneous recombination • Unforeseen solutions by the system? Szostak’s vesicles