Notebook

Week 1&2 7.16-7.29

       Preparation
  1. 1. Research on the methods of introducing mutations;
  2. 2. Research on application direction and field;
  3. 3. Research on screening system;

Week 3&4 7.30-8.12

       Confirmation
  1. 1. Discussion about screening system;
  2. 2. Discussion on methods of building directed evolution machine;
  3. 3. Confirm EvolvR as the continuous directed evolution system
  4. 4. Confirm selecting tagatose and borneol bioproducing as the field to continuously and directly evolve;
  5. 5. Confirm toxicity as the screening system

Week 5,6&7 8.12-9.1

       Plasmid construction
  1. 1. Construct the target plasmids by homologous recombination;
  2. 2. Construct the target plasmids(KanMX and PkmEvolevR) through rational design and transfer them into Escherichia coil;
  3. 3. Large-scale culture the transformed bacteria to collect the product;
  4. 4. Extract plasmids from E-coli and Linearize by Endonuclease enzymes;

Week 8&9 9.2-9.15

       Platform construction
  1. 1. Transfer plasmids about enzymes used for tagatose and borneol synthesis into yeasts;
  2. 2. Colony PCR to check the result of transformation;
  3. 3. Rationally design on site-directed mutagenesis;

Week 10,11-- 9.16-10.3

       Assembly
  1. 1. Transfer KanMX and PkmEvolevR into yeast cells to construct the continuous directed evolution system;
  2. 2. Colony PCR to check the result of transformation;