Team:Osaka/week10
From 2010.igem.org
September 26 (Sun)
- Transfer to culture solution (yesterday's transformations)
- Miniprep of 1-6N, 2-2F, 1-6I, 021 faint hint of red detected
- Restriction digest
- 1-6N, 1-6I with EcoRI, SpeI
- 2-2F, 021 with XbaI, PstI
- Gel electrophoresis
- (RESULTS?)
- Miniprep of parts moved to solution culture this morning
- Cut check with XbaI, PstI <- ??these are not biobrick plasmids!
- Cel8 - ok
- Cel44 - ??
- Man26 - ok
- Xyn10 - ??
- 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)
- Transformation of ligation products
- Transfer of yesterday's transformations to solution culture: 001-2, 025
September 27 (Mon)
- PCR of Man26, CelB
- PCR of Man48 (??), CBM from XynAcc
- 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
- Gel electrophoresis
- 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 (" ")
- Transformation of ligation products
- Transfer to solution culture: 026, 027, 028, 029, 030, 031
September 28 (Tue)
- Miniprep of 026, 027, 028, 030, 031
- Restriction digest
- 026, 028, 031 with XbaI, PstI
- 027, 030 with EcoRI, SpeI
- Gel electrophoresis
- (RESULTS?)
- Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
- Transformation of ligation products as well as 3-2P
ID | Part Name | Resistance | Description |
---|---|---|---|
3-2P | <bbpart>BBa_</bbpart> | A | ?? |
September 29 (Wed)
- Transfer to solution culture 025, 026, 027, 028, 030, 031
- 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)
- 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?)
- PCR
- pgsB - cloning from plasmid
- pgsB - amplification from 021
- Man28 - amplification from previous product
- PCR purification of products
- PCR of CelB, XynA-CBM
- Miniprep of cultures inoculated this morning (total culture time: 12hr)
- 025 -> ok (colorless)
- 027 -> turned red; discarded
- others: ok?
- Restriction digest of 026, 028, 031 with XbaI, PstI
- Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
- Gel electrophoresis of PCR products
- Restriction digests:
- pgsB, Man (??) with EcoRI, SpeI
- today's miniprepped plasmids with EcoRI
- 1-1C with EcoRI, SpeI
September 30 (Thu)
- PCR cloning
- CM10
- CelB (repeat)
- ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
- glr (repeat)
- PCR purification: CM10, XynA-CBM
- Restriction digest of all above PCR products with EcoRI, SpeI
- Gel electrophoresis
- 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
- Transformation of ligation products
- PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
- Gel electrophoresis
- (RESULTS?)
- Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
- Restriction digest of miniprepped parts
- 004, 005, 032 with EcoRI, SpeI (same as earlier today)
- 033, 3-2P (same as on 9/29)
- Gel electrophoresis
- (RESULTS?)
October 1 (Fri)
- Gel electrophoresis (?)
- 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
- Transformation of ligation products
- PCR of ENO2 promoter, ADH2 promoter, SUC2 signal sequence
- Parts from NYU team:
ID | Part Name | Resistance | Description |
---|---|---|---|
R1 | <bbpart>BBa_K416003</bbpart> | A,K | yeast secretion tag |
R2 | <bbpart>BBa_K416004</bbpart> | A | Aga2 yeast cell surface display tag with linker |
- Miniprep of 026, 028, 030
- Restriction digest
- 026, 028 with XbaI, PstI
- 030 with EcoRI, SpeI or EcoRI only
- Gel electrophoresis
- (RESULTS?)
- Transfer to solution culture: 021, 025, 026, 034, 035
October 2 (Sat)
- Restriction digest of plasmid backbones: 1-5A, 1-3A with XbaI, PstI
- Electrophoresis & purification from gel
- 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?)
- PCR cloning from yeast genome ENO2, ADH2 promoters
- Transfer to culture solution: 006, 007, 010, 011, 036, 037, 038
- Miniprep of 021, 025, 026, 034, 035 followed by restriction digest with XbaI, PstI
- Gel electrophoresis
- 021 - OK
- 025 - OK
- 026 - OK
- 034 - bad
- 035 - OK