Team:Osaka/Notebook
From 2010.igem.org
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Notebook
July 29 (Thu)
Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura
- Safety lecture for junior members.
- Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).
July 31 (Sat)
Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka
- Meeting
- Summer project schedule
- List of genes to clone
August 2 (Mon)
Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka
- Culture medium preparation
- LB agar plates (49 antibiotic-less plates)
- LB liquid medium (500 ml)
- Competent cells preparation - Nojima Method
- SOB medium (MgCl2 not yet added) -> stored at 4˚C
- TB buffer -> stored at 4˚C
- Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n
Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.
August 3 (Tue)
Attendance: Nakamura, Kakuda, Saka, Yasumoto, Teoh
- Competent cells preparation (continued)
- Preparation of glucose solution for making SOC medium.
- Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
- (Night) Transfer from pre-culture to growth culture.
August 4 (Wed)
Attendance: Nakamura, Saka, Kakuda, Yasumoto, Torigata, Teoh
- OD measurements throughout the day till required OD (0.3~0.7) was obtained.
- Completion of competent cells according to protocol.
August 5 (Thu)
Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino
- Transformation of Registry parts (See Table 1).
Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.
ID | Part Name | Resistance | Description |
---|---|---|---|
2-20J | <bbpart>BBa_K118023</bbpart> | A | C. fermi endocellulase Cen A coding |
2-20H | <bbpart>BBa_K118022</bbpart> | A | C. fermi exocellulase Cex coding |
1-2M | <bbpart>BBa_B0034</bbpart> | A | RBS |
1-13D | <bbpart>BBa_B0010</bbpart> | A | terminator |
1-1D | <bbpart>BBa_R0010</bbpart> | A | promoter |
1-18F | <bbpart>BBa_E1010</bbpart> | K | RFP coding |
August 6 (Fri)
Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh
- Colony check
- All transformed cells produced colonies!
- Non-transformed cells (negative controls) did not grow on Amp, Kan or Cam plates -> confirmed lack of natural antibiotic resistance
- Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C
August 7 (Sat)
Attendance: Nakamura, Saka, Yasumoto, Takino
- Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
- Transformation of construction plasmids (See Table 2)
- Meeting
ID | Part Name | Resistance | Description |
---|---|---|---|
1-1C | <bbpart>pSB1A3</bbpart> | A | construction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>) |
1-3A | <bbpart>pSB1C3</bbpart> | C | (" ") |
1-5A | <bbpart>pSB1K3</bbpart> | K | (" ") |
August 8 (Sun)
Attendance: Nakamura, Yasumoto
- Colony check
- All parts successfully transformed
- Transfer to LB culture medium
August 9 (Mon)
- Miniprep of 1-1C
- Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
- (WHICH ENZYMES?)
- Gel electrophoresis of digests ("cut check")
- 2-20H, 2-20J, 1-1C -> OK
- 1-1D, 1-18F, 1-13D, 1-2M -> not cut (single band, MW approx. equal to vector + insert)
- Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
- Yesterday's inoculated culture mediums contained the wrong antibiotics!
- Transformation of <bbpart>BBa_I13521</bbpart>, <bbpart>BBa_I13522</bbpart>, <bbpart>BBa_I13600</bbpart>, <bbpart>BBa_K204031</bbpart>, <bbpart>BBa_K204051</bbpart>, <bbpart>BBa_K204032</bbpart> from last year's stock
August 10 (Tue)
- Miniprep of 1-3A, 1-5A
- Restriction digests of 1-3A, 1-5A
- Gel electrophoresis
- 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
- 1-3A, 1-5A -> OK; others -> not cut (again)
- 2nd run: 1-1D, 1-18F, 1-13D, 1-2M (2nd repeat)
- all parts not cut
- 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
August 11 (Wed)
- Miniprep of last year's parts transformed on Monday
- Gel electrophoresis of 1-1D, 1-18F, 1-13D, 1-2M (3rd repeat!!!)
- all 4 not cut... AGAIN
- so far all restriction digests involving XbaI seem to have failed; problem with enzyme stock?
- will try with different set of restriction enzymes next week
- Sent miniprepped last year's parts to ECUST team in Shanghai, China
August 16 (Mon)
- Restriction digests
- 1-13D, 1-2M, 1-1D, 1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
- 2-20J with XbaI, PstI to check/confirm XbaI activity
- Gel electrophoresis of digests
- (RESULTS?)
- Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D, 1-2M, 1-1D, 1-18F
- 3ml LB liquid medium; 3μl Amp or 0.6μl Kan solution added
August 17 (Tue)
- Mini-prep of 1-13D, 1-2M, 1-1D, 1-18F parts inoculated yesterday (2 of each)
- 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
- 1-1D, 1-18F with EcoRI, SpeI
- 1-13D, 1-2M with XbaI, PstI
- (RESULTS?)
- Transformation of secretion tag parts using 25μl of competent cells each(See Table 3)
ID | Part Name | Resistance | Description |
---|---|---|---|
2-22P | <bbpart>BBa_K103006</bbpart> | A | OmpA outer membrane protein + linker |
1-2J | <bbpart>BBa_J32015</bbpart> | A,K | PelB leader sequence |
August 18 (Wed)
iGEM Japan Meet-Up in Kyoto Attendance: Nakamura, Yasumoto, Saka, Kakuda
August 19 (Thu)
- Transfer of 2-22P, 1-2J to solution culture
- Gel electrophoresis of digests from 'cut check' products from Tuesday
- repeat run, but each digest together with undigested plasmid DNA)
- 2% agarose gel instead of the usual 1%
- (RESULTS?)
- Gel electrophoresis of 1-1D digest only
- (RESULT?)
- Multiple restriction digests of 1-1D to check for problems at restriction sites
- tried the following: EcoRI only; SpeI only; EcoRI + SpeI
- Night: miniprep of 2-22P, 1-2J inoculated in the morning
August 20 (Fri)
- Gel electrophoresis of 1-1D and its digests
- (RESULTS?)
- 'Cut check' of parts miniprepped the night before
- both 2-22P & 1-2J cut with XbaI, PstI
- enzyme inactivation at 80°C, 20min
- (RESULTS?)
- Restriction digest of 2-20J (WHICH ENZYMES?)
- Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
- reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
- reaction at room temperature for 10min; ligase inactivation at 80°C for 20min
- Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix
August 21 (Sat)
- Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
- we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded
- 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
- ligation product designated as 001; Chloramphenicol resistance
- same ligation mix composition as yesterday's
- Transformation of 001 with pre-incubation for 1.5hr instead of 1hr
August 22 (Sun)
- Transfer of 001 to culture solution; incubation at 30°C (why??)
- Transformation of the following parts (See Table 4)
- O/N incubation at 37°C as per normal
ID | Part Name | Resistance | Description |
---|---|---|---|
2-4A | <bbpart>BBa_J63005</bbpart> | A | yeast ADH1 promoter |
F1 | N/A | A | beta-galactosidase from Edinburgh team |
F2 | N/A | C | RBS + F1 |
F3 | N/A | C | Lac promoter + RBS + F1 |
August 23 (Mon)
- Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
- Miniprep & 'Cut check' of 001
- cut at EcoRI, SpeI
- gel run with DNA ladder, digested plasmid, undigested plasmid
- (RESULTS?)
- Transfer of 3 more colonies of 001 to liquid solution (to store as glycerol stock - see Tue notes)
- Transformation of the following registry parts (See Table5)
ID | Part Name | Resistance | Description |
---|---|---|---|
2-2O | <bbpart>J63003</bbpart> | A | yeast Kozak sequence |
3-11I | <bbpart>K105027</bbpart> | A | 'cyc100' minimal promoter |
August 24 (Tue)
- Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
- transfer to solution culture
- Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
- (RESULTS)
- Transfer of F1 to solution culture (why?)
- Preparation of glycerol stock of cell culture containing 001 (why?)
- 200ml of culture solution mixed with 100ml of 50% glycerol
- stored at -80°C
August 25 (Wed)
- Miniprep of parts in solution culture: 2-2O, 3-11I, F1
- Cut check of 3-11I & F1 with EcoRI, SpeI
- (RESULTS?)
August 26 (Thu)
- Transformation of <bbpart>BBa_K204022</bbpart>, <bbpart>BBa_K204025</bbpart>, and <bbpart>BBa_K204040</bbpart> to send to Shanghai ECUST team in China.
August 27 (Fri)
- Transfer of yesterday's transformed parts (all produced colonies) to solution culture
- Transformation of the following parts (See Table 6)
- using competent cells opened on 8/20
- Preparation of YPD yeast culture medium with the following recipe (See Table 7)
- pH was adjusted to 5.8
- autoclaved before use
- 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
- Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar
ID | Part Name | Resistance | Description |
---|---|---|---|
1-1K | <bbpart>BBa_J63010</bbpart> | A | Protein fusion vector (Silver standard) |
1-1I | <bbpart>BBa_J63009</bbpart> | A | Low copy protein fusion vector (Silver standard) |
3-3G | <bbpart>BBa_K157013</bbpart> | A | 15aa glycine-serine linker (Freiburg standard) |
MiliQ water | 1 liter | |
Bacto tryptone | 20.0g | 2% |
Bacto yeast extract | 10.0g | 1% |
Glucose | 20.0g | 2% |
August 28 (Sat)
- Miniprep of parts in solution culture
- Restriction digest (for cut check) - 37°C for 30min
- 2-4A & 3-11I with EcoRI, SpeI
- 2-2O with XbaI, PstI
- K204022, K204025, K204040 wih EcoRI, PstI
- Gel electrophoresis of digests
- Plasmids not detected for 2-4A & 3-11I - mistake during miniprep? culture duration too long, plasmid loss occurred?
- Transfer of the following parts to solution culture
- 1-1K, 1-1I, 3-3G (yesterday's transformations)
- 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates)
August 29 (Sun)
- Miniprep: 1-1K, 1-1I, 3-3G, 2-4A, 3-11I
- Restriction digests
- for checking: 1-1K, 1-1I, 3-3G with EcoRI, PstI
- for assembly: 2-4A, 3-11I with EcoRI, SpeI (upstream parts)
- Gel electrophoresis for confirmation
- Inserts seem to be present in all samples
- 3A assembly ligations:
- 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as 002
- 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as 003
- 2-2O using XbaI, PstI digest from yesterday
- 1-5A has Kan resistance
- ligation reaction for 10 mins at room temperature followed by 20min inactivation at 80°C
- Transformation of ligation products 002 and 003
August 30 (Mon)
- Restriction digests for 3A assembly
- 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
- 2-20J (CenA), 2-20H (Cex), F1 with XbaI, PstI
- Gel electrophoresis of the digests to confirm inserts
- all OK
- Transfer of 002 and 003 to solution culture (3 colonies each)
August 31 (Tue)
- Miniprep of 002, 003
- Cut check of 002, 003 with EcoRI, SpeI
- 003 was properly cut, but the insert length was inconsistent; looking back at 8/28 gel result, length of 2-2O (downstream part in 003) also seemed to be longer than expected
- Repeat colony pick-up and solution culture of 2-2O (5 colonies this time)
September 1 (Wed)
- Transformation (See Table 7)
- Miniprep of 5 separate cultures of 2-2O inoculated yesterday
- Cut check of 2-2O with XbaI, PstI
- 0.7kbp bands in all 5 samples even though insert is supposed to be only 18bp - problem with the part (inconsistency confirmed from registry info page)
- obtain Kozak sequence by PCR instead?
ID | Part Name | Resistance | Description |
---|---|---|---|
1-12D | <bbpart>BBa_E2030</bbpart> | K | yeast-optimized EYFP |
1-12B | <bbpart>BBa_E2020</bbpart> | K | yeast-optimized ECFP |
3-2K | <bbpart>BBa_K165001</bbpart> | A | yeast GAL1 promoter |
1-7D | <bbpart>BBa_J63006</bbpart> | A | yeast GAL1 promoter + Kozak sequence |
September 2 (Thu)
- Colony check
- 1-12D, 3-2K, 1-7D produced colonies -> inoculated into solution culture
- 1-12B did not transform successfully
- 3A assembly ligations:
- 001 as upstream, 1-2J as downstream, 1-3A as vector; product designated as 004
- 001 as upstream, 2-22P as downstream, 1-3A as vector; product designated as 005
- Transformation of ligation products
September 3 (Fri)
- Colony check: yesterday's transformations seem to have failed; repeat of transformations of 004 and 005 with 50μl competent cells, 2μl ligation product (note: colonies appeared later; these repeats were then discarded)
- Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI
- all lengths ok
- Transfer of yesterday's transformations (colonies appeared later in the evening) to culture solution (2 colonies picked up from each plate)
September 4 (Sat)
- Miniprep of 004, 005
- Restriction digest of 004, 005 and 1-7D (as control) with EcoRI, SpeI
- Gel electrophoresis
- 1-7D -> OK
- 004 -> insert length same as 001; since both upstream part 001 and vector 1-3A were C resistance, 3A assembly must have failed to yield ligation product; try Standard Assembly!
- 005 -> ??
- Gel electrophoresis followed by purification of 001 to isolate insert -> Standard Assembly
- gel purification performed according to protocol in QIAquick Spin Handbook
- Ligation of gel-purified 001 to 1-2J or 2-22P, with vector 1-3A, to make 004 or 005 respectively (same 004 and 005 as designed before)
September 5 (Sun)
- Heat inactivation of yesterday's ligation mixes at 80°C for 20min followed by transformation.
September 6 (Mon)
- Transfer of yesterday's transformations to solution culture
- Transformation of the following registry parts (See Table 8)
ID | Part Name | Resistance | Description |
---|---|---|---|
2-10F | <bbpart>BBa_K081005</bbpart> | A | constitutive promoter from combinatorial library + RBS |
2-10H | <bbpart>BBa_K081006</bbpart> | A | lambda phage promoter + RBS |
September 7 (Tue)
- Miniprep of 004, 005
- Cut check with EcoRI, SpeI
- both insert lengths ok!
- Transformation of DNA for PGA synthesis-related genes (See Table 9)
- Transfer to solution culture
- 004, 005 transformed on 9/5 (pick up from fresh colonies) -> needed more plasmid
- 2-10F, 2-10H transformed yesterday
ID | Part Name | Resistance | Description |
---|---|---|---|
A01 | pTPG01-1 | A | plasmid pTrc99A with pgs genes inserted |
A02 | pTPG01-2 | A | '' |
A03 | pBSGR3 | K | glutamine racemase |
September 8 (Wed)
- Miniprep 2-10F, 2-10H, 004, 005
- Restriction digest of above parts with EcoRI, SpeI; also with only EcoRI as negative control
- Gel electrophoresis
- new batch of EtBr for staining
- (RESULTS?)
- Transfer of A01~A03 to solution culture
- 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
- it took several tries to get a successful reaction
September 9 (Thu)
- Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
- Purification of 9/8 PCR products from gel using QIA Quick Spin gel extraction kit (why not use PCR purification kit??)
- Restriction digest of A01~03 with EcoRI, PCR products with XbaI, PstI
- 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 -> ?
- Another round of PCR to amplify Cex as Silver standard part (why?)
- 10X dilution of template
- 68°C annealing temp
- 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)
- Transformation of newly assembled parts 006~009
- Transfer of 006~009 to solution culture.
September 10 (Fri)
- 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
- Restriction digests of 004 & 005 (9/4 ligations) with EcoRI, SpeI; followed by gel electrophoresis
- (RESULTS?)
- Restriction digests of Fcex (purified today), FcenA (amplified on 9/9) with XbaI, PstI followed by gel electrophoresis
- (RESULTS?)
- 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)
- Transformation of newly assembled parts Fcex, 006, 007, 010, 011.
- Colony check of 9/9 transformations
- 006: no colonies
- 007: no colonies
- 008: >100 colonies bad insert?
- 009: >100 colonies
- FcenA: >100 colonies
- Transfer of 008, 009, FcenA, 001, 2-10F to solution culture. (more 001, 2-10F needed)
September 11 (Sat)
- Miniprep of 008, 009, FcenA, 001, 2-10F.
- Ristriction digest of 008, 009, 001, 2-10F with EcoRI, SpeI and FcenA with XbaI, PstI.
- 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
- Restriction digest of FcenA with EcoRI.
- Gel electrophoresis of FcenA
- FcenA was digested by EcoRI -> O.K.
- 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?
- Transfomation of newly assembled parts 012, 013
- Transfer of Fcex, 006, 007, 010, 011 (transformed yesterday) to solution culture.
September 12 (Sun)
- Miniprep of Fcex, 006, 007, 010, 011
- Restriction digests of 006, 007, 010, 011 with EcoRI, SpeI; Fcex with XbaI, PstI
- Gel electrophoresis of digests
- (RESULTS)
- Ligation for 3A assembly
- 014: 011 as upstream, 1-13D as downstream, 1-3A as vector
- Transformation of 014 using 2μl ligation product with 50μl competent cells
- Moved yesterday's transformation plates (012, 013) to 4°C refrigerator no RFP expression from vector plasmids at all?
- Transfer of A01, A02, A03 (previously transformed) to solution culture
September 13 (Mon)
- Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis
- (RESULTS?)
- 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
- Gel electrophoresis of PCR products
- (RESULTS?)
- Gel purification of PCR product from F1
- Restriction digest of PCR product (F1) and 1-5A, both with EcoRI, SpeI
- Gel electrophoresis of PCR product from A01
- band visible around 400~500bp, which was correct length -> PCR conditions identified!
- Transformation of A01, A02, A03
- 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)
- Miniprep of A02, 012, 013, 014
- Restriction digests
- 012, 013, 014 with EcoRI, PstI
- A02 with EcoRI
- 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
- 1-13D cut check with XbaI, PstI
- 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)
- Transformation of ligated products (012~014)
- 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)
- 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)
- 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)
- Miniprep A02, A03 (E. coli failed to grow in A01)
- Cut check of A02, A03 with EcoRI
- A02 is ok
- Transformation of 3-22G
- (INFO?)
- PCR cloning of pgsC from A02
- Colony check & transfer of cellulase parts from Karita-sensei to solution culture
- Preparation of lysis buffer for yeast genome DNA extraction according to [insert reference]
Component | Volume Added | Final Concentration | Triton X-100 | 100μl | 2% |
---|---|---|---|---|---|
10%SDS | 500μl | 1% | |||
5M NaCl | 100μl | 100mM | |||
20mM Tris-HCl(ph8.0) | 2.5ml | 10mM | |||
0.5M EDTA(ph8.0) | 10μl | 1mM | |||
dH2O | 1790μl | ||||
TOTAL | 5ml |
- PCR to clone pgsA, pgsB by 'Megaprimer' method using NEB Phusion polymerase
September 16 (Thu)
- Gel electrophoresis to verify yesterday's PCR results
- pgsA megaprimer: 750bp -> OK
- pgsB megaprimer: 170bp -> OK
- pgsC: 450bp -> very faint band?
- Gel extraction of pgsA, pgsB megaprimers
- 2nd step of megaprimer PCR for pgsA, pgsB
- failure; possible causes:
- short annealing step?
- low denaturation temperature?
- mistakes in procedure?
- failure; possible causes:
- PCR
- pgsC using correct (redesigned) primers
- pgsA, B megaprimer 2nd step repeat using reaction mix composition modified from OpenWetWare
- gel purification of PCR products
- Miniprep of BglX (temporary designation for part cloned from F1 with faulty primer), Cel5, Cel8, Cel44, Man26, Xyn10
- Cut check of miniprepped parts with XbaI, PstI
- Yeast genome DNA extraction (detailed protocol will be provided elsewhere) according to [reference article]
- Gel electrophoresis
- yeast genome DNA
- PCR products
- pgsC -> OK
- Restriction digest
- pgsC (PCR product) with EcoRI, PstI
- 1-1C, 1-3A, 1-5A (vectors) with EcoRI, SpeI
September 17 (Fri)
- 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?
- PCR of ADH1 terminator (repeat)
- annealing temperature was lowered from 70°C to 60°C
- PCR failed again -> possible RNA contamination?
- 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
- Gel electrophoresis of yesterday's digests: pgsC, 1-1C, 1-3A, 1-5A
- Miniprep of 013 and 3-22G
- 012, 014 culture solutions have turned red -> picked-up colonies had RFP-carrying vectors, not ligated plasmids; discarded!
- Cut check of 3-22G with XbaI, PstI; 013 with EcoRI, PstI
- (RESULTS?)
- 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?)
- PCR cloning of XynA CBM, pgsA
- Colony pick-up & transfer to solution culture: 006, 007 (9/10 ligation/transformation) more plasmids needed?
- Transformation of 004, 005, 008, 009, 018, 019
September 18 (Sat)
- PCR cloning of pgsA by overlap extension megaprimer method doesn't seem to work so well
- Gel electrophoresis of PCR product (after overlap step) of pgsA
- correct band seems to be obtained
- Gel extraction followed by restriction digest of PCR product with EcoRI, PstI
- Miniprep of 006, 007
- Restriction digest with EcoRI, PstI followed by gel electrophoresis
- PCR of pgsB (1st fragment for overlap extension)
- tried 2 times but couldn't get amplification product!
- Transfer to solution culture: 004, 005, 008, 009, 018, 019
September 19 (Sun)
- PCR of pgsB (repeat)
- again, no product :(
- PCR of pgsB (4th attempt, including yesterday's)
- no product
- Miniprep of 004, 005, 008, 009, 018, 019
- Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI
- 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?
- PCR of pgsB 1st fragment (5th attempt)
- (RESULTS?)
- PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension
- PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension
- PCR: Phusion activity check using BglX as template & the primers that generated it
- PCR: pgsB template check using the outermost primers
September 20 (Mon)
- Transfer to solution culture: 004, 005, 006, 007, 008 ,009 ,019
- 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?
- PCR: primer check
- pair of primers for each overlap segment were tested
- (RESULTS?)
- Digestion of pgsA (PCR product) and 1-1C with EcoRI, PstI
- Ligation to make new part 020: pgsA as insert, 1-1C as vector
- Transformation of ligation product
- 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)
- Miniprep of yesterday's cultures: 004, 005, 006, 007, 008 ,009; 019 was discarded (turned red)
- Restriction digest of miniprepped parts with EcoRI, SpeI
- 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?
- PCR to synthesize C-terminal half of pgsB (overlap extension method continued)
- band of correct size obtained!
- PCR cloning of Man26B, CelB
- gel run failed to turn up bands; repeat with lower annealing temp
- repeat run succeeded!
- Inoculated YPD liquid culture medium with yeast
- New part 020 (contains pgsA) transferred to solution culture
- PCR of pgsB (final step - extension of overlapping fragments)
- Gel electrophoresis to extract PCR products (Man26, CelB) as well as 1-5A for plasmid backbone
September 22 (Wed)
- Gel electrophoresis of 1-5A, 1-3A, 1-1C, PCR product (pgsB) followed by extraction/purification
- Restriction digest of extracted parts with EcoRI, SpeI
- Miniprep of 020
- Restriction digest of 020 with XbaI, PstI (for gel run to check & further assembly)
- Restriction digest of pgsC, pgsA with EcoRI, SpeI
- Gel electrophoresis of all digested parts above
- 020 was bad; repeat ligation?
- Transfer of 006, 007 to solution culture (pick up from new colonies?)
- 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
- Transformation of above ligation products
- Extraction of genome DNA from yeast cultured yesterday
- PCR cloning of yeast parts from genomic DNA
- ADH1 terminator
- ADH2 promoter
- CYC1 terminator
- ENO2 promoter
- SUC2 leader sequence
September 23 (Thu)
- Gel electrophoresis of yesterday's PCR products followed by extraction
- Restriction digest of PCR products with EcoRI, SpeI
- ENO2, ADH2 incorrect length -> repeat
- PCR cloning of ENO2 promoter, ADH2 promoter, glr (glutamate racemase)
- Gel electrophoresis of crude PCR product, extraction & purification from gel
- ENO2 promoter, ADH2 promoter, glr obtained!
- PCR cloning of CelB, Man26B, Cel44A (Cel44A: internal mutations needed; 1st step of overlap extension fragment generation)
- Gel electrophoresis of CelB, Man26B, Cel44A PCR products
- (RESULTS?)
- Miniprep of 006, 007 followed by restriction digest with EcoRI, SpeI
- Gel electrophoresis of 006, 007
- (RESULTS?)
- 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?
- no white/non-RFP colonies on 021 (pgsB) plate
September 24 (Fri)
- Inoculation of Karita-sensei's cellulase parts into fresh LB (need more plasmid); also, 024 (beta-glucosidase)
- Extraction from iGEM distribution plates:
ID | Part Name | Resistance | Description |
---|---|---|---|
1-6N | <bbpart>BBa_</bbpart> | A,K | T7 promoter |
2-2F | <bbpart>BBa_</bbpart> | A | T7 polymerase |
1-6I | <bbpart>BBa_</bbpart> | A | tetracycline-repressible promoter |
- PCR purification of yesterday's Cel44A
- Miniprep of 019, 020
- Restriction digest of 019, 020 with XbaI, PstI
- inserts of correct lengths obtained!
- 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 ???
- Transformation of ligation products
- PCR cloning (repeat) of Man26, CelB
- Gel electrophoresis
- (RESULTS?)
September 25 (Sat)
- Miniprep of yesterday's solution cultures (cellulase parts-containing plasmids from Karita-sensei)
- Restriction digest of miniprepped plasmid DNA with XbaI, PstI (??? these are not biobrick plasmids!) & gel electrophoresis
- Transformation of miniprepped parts
- Restriction digests
- 001 with EcoRI, PstI
- K1 (??) with EcoRI, SpeI
- Ligations to transfer 001, K1 into 1-1C (Amp-resistance) vector -> designated as 001-2
- 025: xylanase (K1) in 1-1C vector
- PCR cloning of CelB
- Transformation of 001-2, 025
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 | ?? |
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?)