Team:Osaka/week5

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August 22 (Sun)

  1. Transfer of 001 to culture solution; incubation at 30°C (why??)
  2. Transformation of the following parts (See Table 4)
    • O/N incubation at 37°C as per normal
Table 4
IDPart NameResistanceDescription
2-4ABBa_J63005Ayeast ADH1 promoter
F1N/AAbeta-galactosidase from Edinburgh team
F2N/ACRBS + F1
F3N/ACLac promoter + RBS + F1

August 23 (Mon)

  1. Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
  2. Miniprep & 'Cut check' of 001
    • cut at EcoRI, SpeI
    • gel run with DNA ladder, digested plasmid, undigested plasmid
    • (RESULTS?)
  3. Transfer of 3 more colonies of 001 to liquid solution (to store as glycerol stock - see Tue notes)
  4. Transformation of the following registry parts (See Table5)
Table 5
IDPart NameResistanceDescription
2-2OJ63003Ayeast Kozak sequence
3-11IK105027A'cyc100' minimal promoter

August 24 (Tue)

  1. Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
    • transfer to solution culture
  2. Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
    • (RESULTS)
  3. Transfer of F1 to solution culture (why?)
  4. Preparation of glycerol stock of cell culture containing 001 (why?)
    • 200ml of culture solution mixed with 100ml of 50% glycerol
    • stored at -80°C

August 25 (Wed)

  1. Miniprep of parts in solution culture: 2-2O, 3-11I, F1
  2. Cut check of 3-11I & F1 with EcoRI, SpeI
    • (RESULTS?)

August 26 (Thu)

  1. Transformation of BBa_K204022, BBa_K204025, and BBa_K204040 to send to Shanghai ECUST team in China.

August 27 (Fri)

  1. Transfer of yesterday's transformed parts (all produced colonies) to solution culture
  2. Transformation of the following parts (See Table 6)
    • using competent cells opened on 8/20
  3. Preparation of YPD yeast culture medium with the following recipe (See Table 7)
    • pH was adjusted to 5.8
    • autoclaved before use
    • 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
  4. Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar
Table 6
IDPart NameResistanceDescription
1-1KBBa_J63010AProtein fusion vector (Silver standard)
1-1IBBa_J63009ALow copy protein fusion vector (Silver standard)
3-3GBBa_K157013A15aa glycine-serine linker (Freiburg standard)
Table 7
MiliQ water1 liter
Bacto tryptone20.0g2%
Bacto yeast extract10.0g1%
Glucose20.0g2%

August 28 (Sat)

  1. Miniprep of parts in solution culture
  2. Restriction digest (for cut check) - 37°C for 30min
    • 2-4A & 3-11I with EcoRI, SpeI
    • 2-2O with XbaI, PstI
    • K204022, K204025, K204040 wih EcoRI, PstI
  3. Gel electrophoresis of digests
    • Plasmids not detected for 2-4A & 3-11I - mistake during miniprep? culture duration too long, plasmid loss occurred?
  4. Transfer of the following parts to solution culture
    • 1-1K, 1-1I, 3-3G (yesterday's transformations)
    • 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates)


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