Team:Osaka/Notebook

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Revision as of 09:26, 13 October 2010

Calendar

Notebook

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September 5 (Sun)

1. Heat inactivation of yesterday's ligation mixes at 80°C for 20min followed by transformation.

September 6 (Mon)

1. Transfer of yesterday's transformations to solution culture
2. Transformation of the following registry parts (See Table 8)

September 7 (Tue)

1. Miniprep of 004, 005
2. Cut check with EcoRI, SpeI
   • both insert lengths ok!
3. Transformation of DNA for PGA synthesis-related genes (See Table 9)
4. Transfer to solution culture
   • 004, 005 transformed on 9/5 (pick up from fresh colonies) -> needed more plasmid
   • 2-10F, 2-10H transformed yesterday

September 8 (Wed)

1. Miniprep 2-10F, 2-10H, 004, 005
2. Restriction digest of above parts with EcoRI, SpeI; also with only EcoRI as negative control
3. Gel electrophoresis
   • new batch of EtBr for staining
   • (RESULTS?)
4. Transfer of A01~A03 to solution culture
5. PCR to make Silver standard-compatible parts from 2-20J (CenA) and 2-20J (Cex) based on protocol in Takara Ex Taq polymerase kit
   • it took several tries to get a successful reaction
     • 1st attempt: template DNA was used directly; concentration too high (failure)?
     • 2nd attempt: 100X, 1000X dilutions attempted without success; this time, over-dilution or stringent annealing temp (68°C) may have been culprit?
     • 3rd attempt: 10X dilutions, annealing temp lowered to 65°C -> success!
   • note: reactions were evaluated by gel electrophoresis of crude PCR product - if band appears at approximately correct length then reaction judged as successful
   • special note of thanks to Nakamura who stayed in lab overnight to run the PCRs

September 9 (Thu)

1. Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
2. Purification of 9/8 PCR products from gel using QIA Quick Spin gel extraction kit (why not use PCR purification kit??)
3. Restriction digest of A01~03 with EcoRI, PCR products with XbaI, PstI
4. Gel electrophoresis of digested parts together with 1-5A 1-5A supposed to be receiving vector, but digested at wrong sites
   • CenA PCR product -> OK (Silver-compatible part designated FcenA)
   • Cex PCR product -> ?
5. Another round of PCR to amplify Cex as Silver standard part (why?)
   • 10X dilution of template
   • 68°C annealing temp
6. Ligation
   • FcenA: PCR product with 1-5A as vector (cut/ligated at X, P)
   • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
   • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
   • 008: 3A assembly of 001 (upstream), Cex PCR product (downstream), 1-5A (vector) bad insert?
   • 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
7. Transformation of newly assembled parts 006~009
8. Transfer of 006~009 to solution culture.

September 10 (Fri)

1. Gel electrophoresis of PCR product from Cex and 2-20H (original Cex part) for comparison
   • PCR product seems ok -> purification from gel; Silver-compatible part designated Fcex
2. Restriction digests of 004 & 005 (9/4 ligations) with EcoRI, SpeI; followed by gel electrophoresis
   • (RESULTS?)
3. Restriction digests of Fcex (purified today), FcenA (amplified on 9/9) with　XbaI, PstI followed by gel electrophoresis
   • (RESULTS?)
4. Ligations
   • Fcex: PCR product from Cex with 1-5A as vector (cut/ligated at X, P)
   • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A　(vector); repeat
   • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector); repeat
   • 010: 3A assembly of 004 (upstream), Fcex (downstream), 1-5A (vector)
   • 011: 3A assembly of 005 (upstream), Fcex (downstream), 1-5A (vector)
5. Transformation of newly assembled parts Fcex, 006, 007, 010, 011.
6. Colony check of 9/9 transformations
   • 006: no colonies
   • 007: no colonies
   • 008: >100 colonies bad insert?
   • 009: >100 colonies
   • FcenA: >100 colonies
7. Transfer of 008, 009, FcenA, 001, 2-10F to solution culture. (more 001, 2-10F needed)

September 11 (Sat)

1. Miniprep of 008, 009, FcenA, 001, 2-10F.
2. Ristriction digest of 008, 009, 001, 2-10F with EcoRI,　SpeI and FcenA with XbaI, PstI.
3. Gel electrophoresis of digested 008, 009, FcenA, 001, 2-10F.
   • 008 -> ???
   • 009 -> O.K.
   • add 1-13D as terminator to 008 and 009'
   • FcenA was not digested by XbaI
4. Restriction digest of FcenA with EcoRI.
5. Gel electrophoresis of FcenA
   • FcenA was digested by EcoRI -> O.K.
6. Ligations for 3A assembly
   • 012: 009 (upstream), 1-13D(terminator,　downstream), 1-3A (vector)
   • 013: 008 (upstream), 1-13D(terminator,　downstream), 1-5A (vector) bad insert?
7. Transfomation of newly assembled parts 012, 013
8. Transfer of Fcex, 006, 007, 010, 011 (transformed yesterday) to solution culture.

September 12 (Sun)

1. Miniprep of Fcex, 006, 007, 010, 011
2. Restriction digests of 006, 007, 010, 011 with EcoRI, SpeI; Fcex with XbaI, PstI
3. Gel electrophoresis of digests
   • (RESULTS)
4. Ligation for 3A assembly
   • 014: 011 as upstream, 1-13D as downstream, 1-3A as vector
5. Transformation of 014 using 2μl ligation product with 50μl competent cells
6. Moved yesterday's transformation plates (012, 013) to 4°C refrigerator no RFP expression from vector plasmids at all?
7. Transfer of A01, A02, A03 (previously transformed) to solution culture

September 13 (Mon)

1. Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis
   • (RESULTS?)
2. PCR test
   • re-cloning of beta-glucosidase from F1 (received from Edinburgh team) into Silver standard-compatible format
   • cloning of pgsC from A01
   • note: primers were misdesigned but PCR performed anyway to confirm whether sequences were complementary, and to identify PCR parameters
3. Gel electrophoresis of PCR products
   • (RESULTS?)
4. Gel purification of PCR product from F1
5. Restriction digest of PCR product (F1) and 1-5A, both with EcoRI, SpeI
6. Gel electrophoresis of PCR product from A01
   • band visible around 400~500bp, which was correct length -> PCR conditions identified!
7. Transformation of A01, A02, A03
8. Transfer of 012, 013, 014 to solution culture
   • RFP expressed from vector plasmids on 012, 013 plates during refrigeration period, so selection of colonies with inserts now possible

September 14 (Tue)

1. Miniprep of A02, 012, 013, 014
2. Restriction digests
   • 012, 013, 014 with EcoRI, PstI
   • A02 with EcoRI
3. Gel electrophoresis of digests
   • A02 was discarded (bad size?)
   • 012~014: digestions repeated with 1μl each of EcoRI, PstI (previous digestions were 0.5μl each)
   • gel electrophoresis of repeat digestions again showed bad sizes for all parts
     • 1-13D terminator part is bad? -> cut check of 1-13D
     • failure to inactivate restriction enzymes before ligations? -> re-ligation
4. 1-13D cut check with XbaI, PstI
5. Re-ligation (3A assembly)
   • previously digested 009, 008, 011 inactivated at 80°C for 20min before ligation
   • 012: 009 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
   • 013: 008 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
   • 014: 011 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/12)
6. Transformation of ligated products (012~014)
7. Gel electrophoresis of yesterday's restriction digests of 1-5A and PCR product from F1 followed by their ligation (1-5A as vector, PCR product as insert)
8. Transfer of A01, A02, A03 to LB liquid culture medium supplanted with 25μl of 2M glucose to prevent leaky expression from Lac promoter (may be affecting growth)
9. Received additional cellulase parts from Karita-sensei: Cel5, Xyn10, Cel44, Man26, Cel8
   • plasmid DNA resuspended in 10μl MiliQ water each
   • transformation with 2μl DNA solution

September 15 (Wed)

1. Miniprep A02, A03 (E. coli failed to grow in A01)
2. Cut check of A02, A03 with EcoRI
   • A02 is ok
3. Transformation of 3-22G
   • (INFO?)
4. PCR cloning of pgsC from A02
5. Colony check & transfer of cellulase parts from Karita-sensei to solution culture
6. Preparation of lysis buffer for yeast genome DNA extraction according to [insert reference]

1. PCR to clone pgsA, pgsB by 'Megaprimer' method using NEB Phusion polymerase

September 16 (Thu)

1. Gel electrophoresis to verify yesterday's PCR results
   • pgsA megaprimer: 750bp -> OK
   • pgsB megaprimer: 170bp -> OK
   • pgsC: 450bp -> very faint band?
2. Gel extraction of pgsA, pgsB megaprimers
3. 2nd step of megaprimer PCR for pgsA, pgsB
   • failure; possible causes:
     • short annealing step?
     • low denaturation temperature?
     • mistakes in procedure?
4. PCR
   • pgsC using correct (redesigned) primers
   • pgsA, B megaprimer 2nd step repeat using reaction mix composition modified from OpenWetWare
   • gel purification of PCR products
5. Miniprep of BglX (temporary designation for part cloned from F1 with faulty primer), Cel5, Cel8, Cel44, Man26, Xyn10
6. Cut check of miniprepped parts with XbaI, PstI
7. Yeast genome DNA extraction (detailed protocol will be provided elsewhere) according to [reference article]
8. Gel electrophoresis
   • yeast genome DNA
   • PCR products
     • pgsC -> OK
9. Restriction digest
   • pgsC (PCR product) with EcoRI, PstI
   • 1-1C, 1-3A, 1-5A (vectors) with EcoRI, SpeI

September 17 (Fri)

1. PCR cloning of ADH1 terminator from yeast genome DNA
   • 4 simultaneous attempts with varying template concentration, thermocycle settings & timing of polymerase addition (before or after initial denaturation)
   • gel electrophoresis showed no PCR product obtained for any of the reactions -> annealing temperatures were too stringent?
2. PCR of ADH1 terminator (repeat)
   • annealing temperature was lowered from 70°C to 60°C
   • PCR failed again -> possible RNA contamination?
3. PCR of ADH1 terminator (2nd repeat)
   • genomic DNA treated with RNase (0.1μl added to 1μl genome DNA; incubation at 37°C for 15min) before using as template
4. Gel electrophoresis of yesterday's digests: pgsC, 1-1C, 1-3A, 1-5A
5. Miniprep of 013 and 3-22G
   • 012, 014 culture solutions have turned red -> picked-up colonies had RFP-carrying vectors, not ligated plasmids; discarded!
6. Cut check of 3-22G with XbaI, PstI; 013 with EcoRI, PstI
   • (RESULTS?)
7. Ligations
   • 018: 3A assembly with 004 as upstream, Fcex as downstream, 1-1C as vector
   • 019: pgsC as insert, 1-1C as vector (cut at?)
8. PCR cloning of XynA CBM, pgsA
9. Colony pick-up & transfer to solution culture: 006, 007 (9/10 ligation/transformation) more plasmids needed?
10. Transformation of 004, 005, 008, 009, 018, 019

September 18 (Sat)

1. PCR cloning of pgsA by overlap extension megaprimer method doesn't seem to work so well
2. Gel electrophoresis of PCR product (after overlap step) of pgsA
   • correct band seems to be obtained
3. Gel extraction followed by restriction digest of PCR product with EcoRI, PstI
4. Miniprep of 006, 007
5. Restriction digest with EcoRI, PstI followed by gel electrophoresis
6. PCR of pgsB (1st fragment for overlap extension)
   • tried 2 times but couldn't get amplification product!
7. Transfer to solution culture: 004, 005, 008, 009, 018, 019

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:

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

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?)