Team:Osaka/week10

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September 26 (Sun)

  1. Transfer to culture solution (yesterday's transformations)
  2. Miniprep of 1-6N, 2-2F, 1-6I, 021 faint hint of red detected
  3. Restriction digest
    • 1-6N, 1-6I with EcoRI, SpeI
    • 2-2F, 021 with XbaI, PstI
  4. Gel electrophoresis
    • (RESULTS?)
  5. Miniprep of parts moved to solution culture this morning
  6. Cut check with XbaI, PstI <- ??these are not biobrick plasmids!
    • Cel8 - ok
    • Cel44 - ??
    • Man26 - ok
    • Xyn10 - ??
  7. Ligations of 9/23 restriction digests (ADH1, ADH2, CYC1, ENO2, SUC2, glr) to 1-1C plasmid backbone digested at E, S sites
    • 026 - ADH1 terminator
    • 027 - ADH2 promoter
    • 028 - CYC1 terminator
    • 029 - ENO2 promoter
    • 030 - SUC2 leader sequence
    • 031 - glr (glutamate racemase)
  8. Transformation of ligation products
  9. Transfer of yesterday's transformations to solution culture: 001-2, 025

September 27 (Mon)

  1. PCR of Man26, CelB
  2. PCR of Man48 (??), CBM from XynAcc
  3. Restriction digests
    • 1-2M with EcoRI, SpeI for assembly later
    • Cel8, Cel44, Cel5 with EcoRI; Xyn10 with PstI (for checking?)
    • 001-2 with EcoRI, SpeI
    • 025 with XbaI, PstI
  4. Gel electrophoresis
  5. Ligations
    • 032: 1-2M as upstream, 019 as downstream, 1-3A as vector
    • 033: 1-2M as upstream, 020 as downstream, 1-3A as vector
    • 004: 001-2 as upstream, 1-2J as downstream, 1-3A as vector (remake using 001-2)
    • 005: 001-2 as upstream, 2-22P as downstream, 1-3A as vector (" ")
  6. Transformation of ligation products
  7. Transfer to solution culture: 026, 027, 028, 029, 030, 031

September 28 (Tue)

  1. Miniprep of 026, 027, 028, 030, 031
  2. Restriction digest
    • 026, 028, 031 with XbaI, PstI
    • 027, 030 with EcoRI, SpeI
  3. Gel electrophoresis
    • (RESULTS?)
  4. Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
  5. Transformation of ligation products as well as 3-2P
IDPart NameResistanceDescription
3-2P<bbpart>BBa_</bbpart>A??

September 29 (Wed)

  1. Transfer to solution culture 025, 026, 027, 028, 030, 031
  2. Ligations (repeat of 9/26 with different dilution of 1-1C)
    • 026 - ADH1 terminator
    • 027 - ADH2 promoter
    • 028 - CYC1 terminator
    • 029 - ENO2 promoter
    • 030 - SUC2 leader sequence
    • 031 - glr (glutamate racemase)
  3. Transformation of 025, 026, 027, 028, 030, 031 (repeat just in case; previous plates almost all red colonies; cannot pick up colony with correct insert?)
  1. PCR
    • pgsB - cloning from plasmid
    • pgsB - amplification from 021
    • Man28 - amplification from previous product
  2. PCR purification of products
  1. PCR of CelB, XynA-CBM
  1. Miniprep of cultures inoculated this morning (total culture time: 12hr)
    • 025 -> ok (colorless)
    • 027 -> turned red; discarded
    • others: ok?
  2. Restriction digest of 026, 028, 031 with XbaI, PstI
  3. Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
  4. Gel electrophoresis of PCR products
  1. Restriction digests:
    • pgsB, Man (??) with EcoRI, SpeI
    • today's miniprepped plasmids with EcoRI
    • 1-1C with EcoRI, SpeI

September 30 (Thu)

  1. PCR cloning
    • CM10
    • CelB (repeat)
    • ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
    • glr (repeat)
  2. PCR purification: CM10, XynA-CBM
  3. Restriction digest of all above PCR products with EcoRI, SpeI
  4. Gel electrophoresis
  1. Ligation of PCR products to 1-1C backbone
    • 021: pgsB
    • 025: XynA-CBM
    • 026: ADH1 terminator
    • 028: CYC1 terminator
    • 031: glr
    • 034: Man
    • 035: CM10
  2. Transformation of ligation products
  1. PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
  2. Gel electrophoresis
    • (RESULTS?)
  1. Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
  2. Restriction digest of miniprepped parts
    • 004, 005, 032 with EcoRI, SpeI (same as earlier today)
    • 033, 3-2P (same as on 9/29)
  3. Gel electrophoresis
    • (RESULTS?)

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)

October 5 (tue)

  1. PCR of glf, Man, ADH2, ENO2, and Gel electrophoresis.
  2. Transformation of yesterday's ligation products.
  3. PCR of glf.
  4. second time?
  5. Miniprep of 039, 2-4A, 3-11l, R1.
  6. Restriction digest of R1 with XbaI, PstI, and Man, 039, 2-4A, 3-11l with EcoRI, SpeI.
  7. Gel electrophoresis of digested plasmids.
    • R1 OK
    • 039 OK
    • 2-4A OK
    • 3-11l X
    • Man OK
    • glr X
  8. Transfer transformants to solution culture: 1-3A, 1-5A, 1-1C, HlyA, ToRA, GeneIII.


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