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Minigenome Project Jingdong Tian, Ph.D. Church Lab March 20, 2003

Minigenome Project Jingdong Tian, Ph.D. Church Lab March 20, 2003. Project Definition of a Minigenome A minimal gene set sufficient to sustain its self-replication in vitro. ~100 genes, ~100 kb (1 kb/gene), derived from E. coli. Minimal functions of the in vitro genome

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Minigenome Project Jingdong Tian, Ph.D. Church Lab March 20, 2003

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  1. Minigenome Project Jingdong Tian, Ph.D. Church Lab March 20, 2003

  2. Project Definition of a Minigenome • A minimal gene set sufficient to sustain its self-replication in vitro. • ~100 genes, ~100 kb (1 kb/gene), derived from E. coli.

  3. Minimal functions of the in vitro genome Functions Genes Number Replication DNA polymerase and other factors 4+ Transcription RNA Polymerase 1-2 RNA processing enzymes 2+ Translation Ribosomal proteins and RNA 58+3 Translation factors 10+ Aminoacyl-tRNA synthetases (ARSs) 20 (tRNAs) (46) Supply: 4 dNTPs, 4 NTPs, 20 amino acids, energy, chemicals and salts

  4. Replication E. coli replication apparatus: Sequences: oriC, origin, ~245 bp, methylated; terD,A; C,B termination, ~23 bp each, ~200 kb apart Protein factors: DnaA origin recognition and melting DnaB helicase DnaC loading helicase/primase RNA pol unwinding DnaJ? Release of initiation complex DnaK? ‘’ SSB single strand maintenance Gyrase fork movement DnaG priming  Sliding clamp  clamp loading (ATPase) Pol III core catalysis  Holoenzyme dimerization Pol I RNA removal Ligase ligation Tus termination (ter sites binding) Dam Methylase

  5. Alternative replication system 1 – for circular genome Bacteriaphage (ds) rolling-circle replication PA: Origin recognition, nicking, covalent binding, resealing. PB: Helicase (DnaB) loader, lagging strand synthesis.

  6. Alternative replication system 2 – for linear genome

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