Notebook

Week 1&2

       13/5/2022-26/5/2022
  1. 1. Research on the reaction component and enzyme
  2. 2. Research on the methods of introducing mutations
  3. 3. Discussion about the continuous directed evolution system
  4. 4. Research on the methods of irrational design and rational design
  5. 5. Discussion about screening system

Week 3&4

       27/5/2022-9/6/2022
  1. 1. Confirmation for selecting TbsADH as the enzyme to continuously and directly evolve
  2. 2. Confirmation for EvolvR as the continuous directed evolution system
  3. 3. Improve the initial reaction system
  4. 4. Preparation on site-directed mutagenesis

Week 5

       10/6/2022-16/6/2022
  1. 1. Examine the toxicity of NA and NK to yeast
  2. 2. Examine the motility of yeast on solid medium and semisolid medium
  3. 3. Confirm toxicity as the screening system
  4. 4. Rational design on site-directed mutagenesis

Week 6&7

       17/6/2022-30/6/2022
  1. 1. Select the ADH kit to identify the enzyme property
  2. 2. Construct the reaction NK-NA reaction system
  3. 3. Construct the target plasmids by homologous recombination

Week 8&9&10

       1/7/2022-21/7/2022
  1. 1. Construct the target plasmids through rational design and transfer them into Escherichia coil
  2. 2. Construct the wild type plasmid and transfer it into Escherichia coil
  3. 3. Large-scale culture the transformed bacteria to collect the product

Week 11&12

       22/7/2022-4/8/2022
  1. 1. Large-scale expression and purification of the TbsADH
  2. 2. Identify the enzyme property and compare WT with TBR1 in the NK-NA reaction system

Week 13&14&15&16

       5/8/2022-1/9/2022
  1. 1. Construct the target plasmids(r-p426/r-p415/r-YEGAP)
  2. 2. Transfer them into yeast cells to construct the continuous directed evolution system

Week 17&18

       2/9/2022-15/9/2022
  1. 1. Get the gene sequence of GDH from bacillus subtilis and transfer it into the E.coli
  2. 2. Large-scale expression and purification of GDH
  3. 3. Detect the optimum temperature and enzyme property of GDH

Week 19&20

       16/9/2022-29/9/2022
  1. 1. Construct the continuous directed evolution system
  2. 2. Screen the highest motility of the yeast and identify the enzyme property