Team:Osaka/Notebook

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Calendar

July
Week S M T W T F S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
week1 25 26 27 28 29 30 31
August
Week S M T W T F S
week2 1 2 3 4 5 6 7
week3 8 9 10 11 12 13 14
week4 15 16 17 18 19 20 21
week5 22 23 24 25 26 27 28
week6 29 30 31
September
Week S M T W T F S
week6 1 2 3 4
week7 5 6 7 8 9 10 11
week8 12 13 14 15 16 17 18
week9 19 20 21 22 23 24 25
week10 26 27 28 29 30
October
Week S M T W T F S
week10 1 2
week11 3 4 5 6 7 8 9
week12 10 11 12 13 14 15 16
week13 17 18 19 20 21 22 23
week14 24 25 26 27 28 29 30
week15 31
November
Week S M T W T F S
week15 1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30










Notebook

October 1 (Fri)

  1. Gel electrophoresis (?)
  2. Ligations
    • 006: 004 as upstream, FcenA as downstream, 1-5A as vector
    • 007: 005 as upstream, FcenA as downstream, 1-5A as vector
    • 010: 004 as upstream, Fcex as downstream, 1-5A as vector
    • 011: 005 as upstream, Fcex as downstream, 1-5A as vector
    • 036: 032 as upstream, 033 as downstream, 1-5A as vector
    • 037: 004 as upstream, 024 as downstream, 1-1C as vector
    • 038: 005 as upstream, 024 as downstream, 1-1C as vector
  3. Transformation of ligation products
  4. PCR of ENO2 promoter, ADH2 promoter, SUC2 signal sequence
  5. Parts from NYU team:
IDPart NameResistanceDescription
R1<bbpart>BBa_K416003</bbpart>A,Kyeast secretion tag
R2<bbpart>BBa_K416004</bbpart>AAga2 yeast cell surface display tag with linker
  1. Miniprep of 026, 028, 030
  2. Restriction digest
    • 026, 028 with XbaI, PstI
    • 030 with EcoRI, SpeI or EcoRI only
  3. Gel electrophoresis
  4. (RESULTS?)
  5. Transfer to solution culture: 021, 025, 026, 034, 035

October 2 (Sat)

  1. Restriction digest of plasmid backbones: 1-5A, 1-3A with XbaI, PstI
  2. Electrophoresis & purification from gel
  3. PCR of SUC2 signal sequence
    • primers diluted with MiliQ water from 100μM to 5μM
    • Taq polymerase added before starting (as opposed to after initial denaturation step)
    • gel run to check -> (RESULTS?)
  4. PCR cloning from yeast genome ENO2, ADH2 promoters
  5. Transfer to culture solution: 006, 007, 010, 011, 036, 037, 038
  6. Miniprep of 021, 025, 026, 034, 035 followed by restriction digest with XbaI, PstI
  7. Gel electrophoresis
    • 021 - OK
    • 025 - OK
    • 026 - OK
    • 034 - bad
    • 035 - OK

October 3 (Sun)

  1. Miniprep of 006, 007, 010, 011, 036, 037, 038 followed by restriction digest with XbaI, PstI
  2. Restriction digest of yesterday's minipreps: 034, 035, 036 with EcoRI, SpeI
  3. Gel electrophoresis
    • 006 - ok
    • 007 - ?
    • 010 - bad
    • 011 - bad
    • 034 - bad
    • 035 - ok
    • 036 - ok
    • 037 - ?
    • 038 - not sufficiently digested?
  4. Another round of restriction digest:
    • spare samples of 010, 011, 037 with XbaI, PstI as above (2 colonies were cultured in solution & miniprepped)
    • 038: more XbaI, PstI added to previous tube
  5. 1-2J, 1-18F, 007, 037 with EcoRI, PstI
  6. Gel electrophoresis
    • (RESULTS?)
  7. Polyacrylamide gel electrophoresis of SUC2 PCR product & elution (recipe/protocol elsewhere)
    • elution buffer added & overnight shaking incubation at 37°C
  8. Cut check of R1, R2 with EcoRI, SpeI
  9. PCR cloning from yeast genome ADH2, ENO2 again
  10. Ligation & transformation:
    • 039: 001 as upstream, 021 as downstream, 1-5A as vector
  11. Cut check of PCR products
    • results bad; repeat!
  12. PCR cloning from yeast genome: ADH2 promoter

October 4 (Mon)

  1. Gel electrophoresis
    • sample?
  2. Transformation og new genes arrived from Utha University.
    • HlyA pSB3K3 19.3mg/ml
    • ToRA pSB3K3 12.9mg/l
    • GeneIII pSB1AK3 197.7mg/ml
  3. Transfer to soltion culture: 039.
  4. Restriction digest of 007 with EcoRI.
  5. Gel electrophoresis: 007.
  6. Restriction digest of eluted SUC with EcoRI and SpeI.
  7. Ligation
    • 008(K): 001(upstream), 2-20H(downstream), 1-5A(vector)
    • 009(K): 001(upstream), 2-20J(downstream), 1-5A(vector)
    • 010(K): 004(upstream), Fcex(023, downstream), 1-5A(vector)
    • 015(C): 001(upstream), 1-13D(downstream), 1-3A(vector)
    • 016(C): 007(upstream), 1-13D(downstream), 1-3A(vector)
    • 044(C): 001-2(upstream), F1(downstream), 1-3A(vector)
    • 045(A): 005(upstream), 024(beta-gluctosidase, downstream), 1-1C(vector)
    • 046(A): 004(upstream), 024(downstream), 1-1C(vector)

TO CHECK/CONFIRM

  • 'K1' (mentioned on 9/250 -> where did it come from?
  • 9/25 - miniprepped parts (inoculated 9/24) were plasmids from karita sensei? biobrick parts pcr-cloned on 9/23? why where these parts transformed immediately after miniprep?
  • 9/24~27 'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)

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