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:
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 |
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.
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
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 | (" ") |
- Meeting
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
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:
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 |
- O/N incubation at 37°C as per normal
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
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
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) |
- using competent cells opened on 8/20
- Preparation of YPD yeast culture medium with the following recipe:
MiliQ water | 1 liter | |
Bacto tryptone | 20.0g | 2% |
Bacto yeast extract | 10.0g | 1% |
Glucose | 20.0g | 2% |
- 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
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 (BglX) with XbaI, PstI
- beta-glucosidase received from Edinburgh team temporarily designated BglX
- 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
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 |
- 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?
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:
ID | Part Name | Resistance | Description |
---|---|---|---|
2-10F | <bbpart>BBa_K081005</bbpart> | A | constitutive promoter + 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
ID | Part Name | Resistance | Description |
---|---|---|---|
A01 | pTPG01-1 | A | plasmid pTrc99A with pgs genes inserted |
A02 | pTPG01-2 | A | '' |
A03 | pBSGR3 | K | glutamine racemase |
- 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)
- 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?)
- Tranfer of A01~A03 to solution culture
- PCR to make Silver standard-compatible parts from 2-20J (CenA), 2-20J (Cex), F1 (BglX) 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 PCR product from gel using QIA Quick Spin gel extraction kit (why not use PCR purification kit??)
- Restriction digest of PCR products with XbaI, PstI
- Gel electrophoresis of digested A01, A02, A03, FcenA, Fcex, 1-5A <- when was this digested with X, P?
- Another round of PCR to amplify Cex
- 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), Fcex (downstream), 1-5A (vector)
- 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
- Transformation of newly assembled parts 006~009
September 10 (Fri)
- Gel electrophoresis of amplified cex, and 2-20H.
- Gel purification of amplified cex.
- Restriction digest Fcex plasmid, FcenA plasmid with XbaI, PstI and 004, 005 with EcoRI, SpeI.
- Gel electrophoresis of digested 004, 005 and Fcex, FcenA, PCR product(FcenA).
- The result of transformation in 9/9
- 008 more than 100 colonies
- 009 more than 100 colonies
- FcenA more than 100 colonies
- 006 none
- 007 none
- Ligation
- 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
- 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
- Fcex: Fcex with 1-5A as vector (cut/ligated at X, P)
- 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 006, 007, Fcex, 010, 011.
- Transfer of 008, 009, FcenA, 001, 2-10F to solution culture.