August 16 (Mon)

  1. Restriction digests
    • 1-13D, 1-2M, 1-1D, 1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
    • 2-20J with XbaI, PstI to check/confirm XbaI activity
  2. Gel electrophoresis of digests
    • (RESULTS?)
  3. Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D, 1-2M, 1-1D, 1-18F
    • 3ml LB liquid medium; 3μl Amp or 0.6μl Kan solution added

August 17 (Tue)

  1. Mini-prep of 1-13D, 1-2M, 1-1D, 1-18F parts inoculated yesterday (2 of each)
  2. 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
    • 1-1D, 1-18F with EcoRI, SpeI
    • 1-13D, 1-2M with XbaI, PstI
    • (RESULTS?)
  3. Transformation of secretion tag parts using 25μl of competent cells each(See Table 3)
Table 3
IDPart NameResistanceDescription
2-22PBBa_K103006AOmpA outer membrane protein + linker
1-2JBBa_J32015A,KPelB leader sequence

August 18 (Wed)

iGEM Japan Meet-Up in Kyoto Attendance: Nakamura, Yasumoto, Saka, Kakuda

August 19 (Thu)

  1. Transfer of 2-22P, 1-2J to solution culture
  2. Gel electrophoresis of digests from 'cut check' products from Tuesday
    • repeat run, but each digest together with undigested plasmid DNA)
    • 2% agarose gel instead of the usual 1%
    • (RESULTS?)
  3. Gel electrophoresis of 1-1D digest only
    • (RESULT?)
  4. Multiple restriction digests of 1-1D to check for problems at restriction sites
    • tried the following: EcoRI only; SpeI only; EcoRI + SpeI
  5. Night: miniprep of 2-22P, 1-2J inoculated in the morning

August 20 (Fri)

  1. Gel electrophoresis of 1-1D and its digests
    • (RESULTS?)
  2. 'Cut check' of parts miniprepped the night before
    • both 2-22P & 1-2J cut with XbaI, PstI
    • enzyme inactivation at 80°C, 20min
    • (RESULTS?)
  3. Restriction digest of 2-20J (WHICH ENZYMES?)
  4. Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
    • reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
    • reaction at room temperature for 10min; ligase inactivation at 80°C for 20min
  5. Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix

August 21 (Sat)

  1. Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
    • we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded
  2. 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
    • ligation product designated as 001; Chloramphenicol resistance
    • same ligation mix composition as yesterday's
  3. Transformation of 001 with pre-incubation for 1.5hr instead of 1hr

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