Team:Osaka/Notebook

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September
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Notebook

September 19 (Sun)

  1. PCR of pgsB (repeat)
    • again, no product :(
  2. PCR of pgsB (4th attempt, including yesterday's)
    • no product
  3. Miniprep of 004, 005, 008, 009, 018, 019
  4. Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI
  5. Gel electrophoresis of pgsA, 004, 005, 008, 009, 018, 019
    • apart from 018 & 019, all are parts digested before; used here to compare with new parts 018, 019
    • (RESULTS?)
    • problem with 019?
  6. PCR of pgsB 1st fragment (5th attempt)
    • (RESULTS?)
  7. PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension
  8. PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension
  9. PCR: Phusion activity check using BglX as template & the primers that generated it
  10. PCR: pgsB template check using the outermost primers

September 20 (Mon)

  1. Transfer to solution culture: 004, 005, 006, 007, 008 ,009 ,019
  2. PCR: Phusion polymerase & template checks (repeat of 9/19?)
    • positive control (BglX) was amplified -> Phusion polymerase seems to be working ok
    • problem with template? primer? thermocycle settings?
  3. PCR: primer check
    • pair of primers for each overlap segment were tested
    • (RESULTS?)　
  4. Digestion of pgsA (PCR product) and 1-1C with EcoRI, PstI
  5. Ligation to make new part 020: pgsA as insert, 1-1C as vector
  6. Transformation of ligation product
  7. PCR of pgsB 1st fragment (n-th repeat??)
  • Note: MANY MANY rounds of PCR carried out today; due to time constraints they are not described here in detail

September 21 (Tue)

  1. Miniprep of yesterday's cultures: 004, 005, 006, 007, 008 ,009; 019 was discarded (turned red)
  2. Restriction digest of miniprepped parts with EcoRI, SpeI
  3. Gel electrophoresis of digested parts 004, 005, 006, 007, 008, 009, 019 (?)
    • 005 - OK
    • 014 - no band visible
    • 019 - bad length - repeat ligation
    • 006, 007 - bad lengths; repeat colony pick-up & culture?
  4. PCR to synthesize C-terminal half of pgsB (overlap extension method continued)
    • band of correct size obtained!
  5. PCR cloning of Man26B, CelB
    • gel run failed to turn up bands; repeat with lower annealing temp
    • repeat run succeeded!
  6. Inoculated YPD liquid culture medium with yeast
  7. New part 020 (contains pgsA) transferred to solution culture
  8. PCR of pgsB (final step - extension of overlapping fragments)
  9. Gel electrophoresis to extract PCR products (Man26, CelB) as well as 1-5A for plasmid backbone

September 22 (Wed)

  1. Gel electrophoresis of 1-5A, 1-3A, 1-1C, PCR product (pgsB) followed by extraction/purification
  2. Restriction digest of extracted parts with EcoRI, SpeI
  3. Miniprep of 020
  4. Restriction digest of 020 with XbaI, PstI (for gel run to check & further assembly)
  5. Restriction digest of pgsC, pgsA with EcoRI, SpeI
  6. Gel electrophoresis of all digested parts above
    • 020 was bad; repeat ligation?
  7. Transfer of 006, 007 to solution culture (pick up from new colonies?)
  8. Ligations
    • 019: pgsC (PCR product) into 1-1C vector
    • 020: pgsA (PCR product) into 1-1C vector
    • 021: pgsB (PCR product) into 1-1C vector
    • all using PCR products purified today
  9. Transformation of above ligation products
  10. Extraction of genome DNA from yeast cultured yesterday
  11. PCR cloning of yeast parts from genomic DNA
    • ADH1 terminator
    • ADH2 promoter
    • CYC1 terminator
    • ENO2 promoter
    • SUC2 leader sequence

September 23 (Thu)

  1. Gel electrophoresis of yesterday's PCR products followed by extraction
  2. Restriction digest of PCR products with EcoRI, SpeI
    • ENO2, ADH2 incorrect length -> repeat
  3. PCR cloning of ENO2 promoter, ADH2 promoter, glr (glutamate racemase)
  4. Gel electrophoresis of crude PCR product, extraction & purification from gel
    • ENO2 promoter, ADH2 promoter, glr obtained!
  5. PCR cloning of CelB, Man26B, Cel44A (Cel44A: internal mutations needed; 1st step of overlap extension fragment generation)
  6. Gel electrophoresis of CelB, Man26B, Cel44A PCR products
    • (RESULTS?)
  7. Miniprep of 006, 007 followed by restriction digest with EcoRI, SpeI
  8. Gel electrophoresis of 006, 007
    • (RESULTS?)
  9. Transfer to solution culture: 019, 020
    • no white/non-RFP colonies on 021 (pgsB) plate
      • insert (PCR product) was not digested properly?
      • problem with gel purification?

September 24 (Fri)

  1. Inoculation of Karita-sensei's cellulase parts into fresh LB (need more plasmid); also, 024 (beta-glucosidase)
  2. Extraction from iGEM distribution plates:
IDPart NameResistanceDescription
1-6N<bbpart>BBa_</bbpart>A,KT7 promoter
2-2F<bbpart>BBa_</bbpart>AT7 polymerase
1-6I<bbpart>BBa_</bbpart>Atetracycline-repressible promoter
  1. PCR purification of yesterday's Cel44A
  2. Miniprep of 019, 020
  3. Restriction digest of 019, 020 with XbaI, PstI
    • inserts of correct lengths obtained!
  4. Ligations for 3A assembly
    • 004: 001 as upstream, 1-2J as downstream, 1-3A as vector
    • 005: 001 as upstream, 2-22P as downstream, 1-3A as vector
    • pgsB 10xHC 1-3A ???
  5. Transformation of ligation products
  6. PCR cloning (repeat) of Man26, CelB
  7. Gel electrophoresis
    • (RESULTS?)

September 25 (Sat)

  1. Miniprep of yesterday's solution cultures (cellulase parts-containing plasmids from Karita-sensei)
  2. Restriction digest of miniprepped plasmid DNA with XbaI, PstI (??? these are not biobrick plasmids!) & gel electrophoresis
  3. Transformation of miniprepped parts
  4. Restriction digests
    • 001 with EcoRI, PstI
    • K1 (??) with EcoRI, SpeI
  5. Ligations to transfer 001, K1 into 1-1C (Amp-resistance) vector -> designated as 001-2
    • 025: xylanase (K1) in 1-1C vector
  6. PCR cloning of CelB
  7. Transformation of 001-2, 025


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?)

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?
  • 'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)

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