1. Prepare one 500 ml (to culture Cas12a E. coli)and three 300ml LB(To culture four colonies of GM E. coli with different integrated template sequences*****, each trial requires 25ml, the excess will be on standby)
    Weigh the following reagents with an electronic balance and add them into 4 conical flasks using a spatula in the following sequence:
    Tryptone* 5 g 3 g
    Yeast extract** 2.5 g 1.5 g
    NaCl*** 5 g 3 g
    Suspend with ddH2O**** until the volume reaches 500 ml 300 ml
    *Tryptone is used to provide essential amino acids such as peptides and peptones to the bacteria
    **Yeast Extract provides a range of organic compounds for bacteria growth
    ***Sodium chloride balances osmotic pressure and provides sodium ions for the bacterium
    ****ddH2O: double distilled water
    *****four types of template sequences:
    Name of the sequence Originates from
    16S Helicobacter pylori
    cagA Helicobacter pylori
    invA Salmonella typhimorium
    ipaH Shigella flexneri
  2. Autoclave all LB (lysogeny broth)
    *No need to adjust the pH as both sodium hydroxide and TRIS buffer are usually unnecessary.
  3. Allocate 150ml autoclaved LB into two equal aliquots, each 75ml.
  4. Add 75μl of Kan+ antibiotics into one flask, and 75μl of Amp+ antibiotics into the other (as the volume ratio between LB and antibiotics should be 1000:1). Label the flasks.
  5. Prepare 4 sets of cell culture tubes, each with 3 tubes.
  6. In each group of tubes, allocate 5ml of Amp+ LB into one tube,and 5ml Kan+ LB into the other two.
  7. Inoculate bacterium (Add 50μl of E. coli-containing-medium into each tube, ending up with four tubes with four different types of GM E. coli), and label the date, its antibiotics, and integrated sequence on the tube. The number and types of cell culture tubes are recorded in the table below:
    Integrated sequence Plasmid name Corresponding antibiotics Number of tubes
    Cas12a pET-28a Kan+ 8
    16S pUC57 Amp+ 1
    cagA 1
    invA 1
    ipaH 1
  8. Place all tubes in the shaker rock for 16 hours, allow it to proliferate