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Team:Osaka/Notebook
From 2010.igem.org
Calendar
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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℃, 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℃ 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 <1>; Chloramphenicol resistance
- same ligation mix composition as yesterday's
- Transformation of <1> with pre-incubation for 1.5hr instead of 1hr
August 22 (Sun)
- Transfer of <1> to culture solution; incubation at 30℃ (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℃ as per normal
August 23 (Mon)
- Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
- Miniprep & 'Cut check' of <1>
- cut at EcoRI, SpeI
- gel run with DNA ladder, digested plasmid, undigested plasmid
- (RESULTS?)
- Transfer of 3 more colonies of <1> 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 <1> (why?)
- 200ml of culture solution mixed with 100ml of 50% glycerol
- stored at -80℃
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℃ 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 <2>
- 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as <3>
- 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℃
- Transformation of ligation products <2> and <3>
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 <2> and <3> to solution culture (3 colonies each)
August 31 (Tue)
１．miniprep
2-20-2-4-A ←共に各3つずつ 2-20-3-11I 2-20-3-11I②ペレットが赤かった
☆Lysis Solutionの保存温度、取扱いミスによって今までの収量が悪かったのでは？ きちんと溶けていない状態で冷蔵していた→60℃に入れて溶かした （最初の段階で60℃に入れて溶かすとあるのでおそらく熱には大丈夫！）
New parts! 2-20-2-4-A ・・・ 002 2-20-3-11I ・・・003
002
yeast ADH1 promoter + kozak segnence(RBS for yeast)
003
cyc 100minimal promoter + kozak segnence (RBS for yeast)
２．制限酵素処理
002①,003① プラスミド 2.5μｌ
EcoRI 1μｌ
Spe I 1μｌ 10×NEbuffer2 5μｌ
100×BSA 0.5μｌ H2O 40μｌ
total 50μｌ
37 ℃ incubate 30min
３．電気泳動 dye 2μｌ、サンプル 10μｌ、Ladder 2μｌ
1%Agalose gel、100V 20min
003①d 002①d Ladder
003①cutはできている
インサートが(18+103)bpよりも明らかに大きい
戻って見直すと2-20のインサートの長さがおかしい
ライゲーションはできている
2-20を再度培養しcut checkする
４．2-20の5つのコロニーをそれぞれLB培地に植える LB3ml＋Amp3μl 16:20~
September 20
- PCR
tube5 control - 5xphusion HF Buffer 4μl
- 10mM dNTP 0.4μl
- primer 1μl
- control template DNA 0.8μl
- phusion 0.2μl
- H2O 13.6μl
- total 20μl
thermal cycle - 98℃ 1min
- 98℃ 5sec
- 60℃ 15sec
- 72℃ 2min30sec 25cycle (boldface)
- 72℃ 10min
- 4℃ ∞
tube1 - GC Buffer 4μl
- dNTP 0.4μl
- 10xprimer Mix 2μl
- template (10xA02) 1μl
- phusion 0.2μl
- H2O 12.4μl
- total 20μl
tube2 - HF Buffer 4μl
- dNTP 0.4μl
- 10xprimer Mix 2μl
- template (10xA02) 1μl
- phusion 0.2μl
- MgCl2(1mM) 4μl
- H2O 8.4μl
- total 20μl
thermal cycle - 98℃ 1min
- 98℃ 5sec
- 40℃ 30sec
- 72℃ 50sec 30cycle (boldface)
- 72℃ 10min
- 4℃ ∞
- Control is good.
- Sample is not good.
- problem of template and primer / thermal cycle and condition of reaction?
- PCR for checking primer
- PCR
- HF Buffer 32μｌ
- 10mM dNTP 3.2μｌ
- Template(×10) 8μｌ
- Phusion 1.6μｌ
- DSW 112μｌ
- total 156.8μｌ
(ⅰ)rev4fwd4 (ⅱ)rev3fwd3 (ⅲ)rev2fwd2 (ⅳ)pgsBrev1 Saka 1 3 5 7 Yasumoto 2 4 6 8
PCR（ⅰ） BlockA
5xPhusion HF Buffer 4μｌ 10mM dNTP 0.4μｌ
Template 1μｌ
primer rev4 0.2μｌ
primer fwd4 0.2μｌ
Phusion 0.2μｌ
DSW 14μｌ
total 20μｌthermal cycle - 98℃ 30sec
- 98℃ 10sec
- 65℃ 30sec
- 72℃ 30sec 35cycle(boldface)
- 72℃ 10min
- 4℃ ∞
PCR（ⅱ）
5xPhusion HF Buffer 4μｌ
10mM dNTP 0.4μｌ
Template (x10) 1μｌ
primer rev3 0.2μｌ
primer fwd3 0.2μｌ
Phusion 0.2μｌ
DSW 14μｌ
total 20μｌ
thermal cycle 98℃ 30sec 98℃ 10sec 70℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃ ∞
PCR（ⅲ）
5xPhusion HF Buffer 4μｌ
10mM dNTP 0.4μｌ
Template (x10) 1μｌ
primer rev2 0.2μｌ
primer fwd2 0.2μｌ
Phusion 0.2μｌ
DSW 14μｌ
total 20μｌ
thermal cycle 98℃ 30sec 98℃ 10sec 70℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃ ∞
PCR（ⅳ） BlockB
5xPhusion HF Buffer 4μｌ
10mM dNTP 0.4μｌ
Template 1μｌ
primer pgsB 0.2μｌ
primer rev1 0.2μｌ
Phusion 0.2μｌ
DSW 14μｌ
total 20μｌ
thermal cycle 98℃ 30sec 98℃ 10sec 60℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃ ∞
Check PCR fragments by each primers. 各primerによる断片作成チェック ～primer erratum primerの正誤確認～ 98℃ 30sec 98℃ 10sec
X℃ 30sec
72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃ ∞ ～Mix composition～
5xPhusion HF Buffer 4μｌ
10mM dNTPs 0.4μｌ
Template(×10) 1μｌ
primer 1 0.2μｌ
primer 2 0.2μｌ
primer 3 0.2μｌ
primer 4 0.2μｌ
polymerase 0.2μｌ
DSW 13.6μｌ
total 20μｌ
8. Electrophoresis Apply all sample 全量アプライ(dye 2μｌ、sample 20μｌ、Ladder 10μｌ) 1%Agalose gel、100V 20min
8 7 6 5 4 3 2 1 Ladder
8 , 7→1 6 , 5→2 2 , 1→3
Elution of each sample from gel and purification
3. pgsB 2nd PCR +再々々々 1st PCR
HF Buffer 4μｌ
dNTP 0.4μｌ
primer pgsB 0.2μｌ
rev2 0.2μｌ
tube1 0.2μｌ
tube2 0.2μｌ
Phusion 0.2μｌ
DSW 14.6μｌ
total 20μｌ
protocol1
98℃ 1min
94℃ 15sec
50℃ 30sec
74℃ 30sec 下３つを2cycle
↓flanking primer3
94℃ 15sec
68℃ 30sec
72℃ 1min 上３つを25cycle
72℃ 10min
4℃ ∞
protocol2 tube8 tube7
98℃ 1min
98℃ 10sec
50℃ 30sec
70℃ 30sec 下３つを2cycle
↓Hauking primers
98℃ 10sec
70℃ 30sec
68℃ 1min 上３つを25cycle
72℃ 10min
4℃ ∞
↓ Electropjoresis
4. Electrophoresis dye 2μｌ、Sample 5μｌ、Ladder 4μｌ 1%Agalose gel、100V 20min
PCR1 PCR2 PCR3 Ladder
5. pgsB 1st PCR (part)
Mix composition
5xPhusion HF Buffer 4μｌ
10mM dNTPs 0.4μｌ
Template 1μｌ
primer rev 0.2μｌ
primer fwd 0.2μｌ
phusion 0.2μｌ
DSW 14μｌ
total 20μｌ
thermal cycle 98℃ 30sec 98℃ 10sec
74℃ 30sec
72℃ 10sec 35cycle(boldface) 72℃ 10min 4℃ ∞
6. To liquid culture
004(C), 005(C) ,006(K) ,007(K) 008(K) ,009(K) ,019(A)
To LB medium 37 ℃ incubate 18:20~
New parts 020(A)
pgsA
7. Ligation
pgsA (Eco,Pst)d 2μl
1-1C (Eco,Pst)d 2μl
10×T4 DNA ligase Buffer 2μｌ
T4 DNA ligase 1μｌ
H2O 13μｌ
total 20μｌ
room temp 10min Ligation
80℃ deactivtion by heat 20min
8. Transformation Competent cell②50μl＋LigationLigation product 2μl Inoculte A plate and incubt at 37oC 2：00～
9. Electrophoresis All PCR product＋dye 2μｌ、Ladder 10μｌ 1%Agalose gel、100V 25min
tube8 tube7 tube6 tube5 Ladder tube2 tube1 Ladder
tube1, 2 は300bpあたり tube5, 6, 7, 8 は740bpあたりのバンドを切り出し gel purification
10. Electrophoresis to check gel purification dye 2μｌ、sample 10μｌ、Ladder 10μｌ 1%Agalose gel、100V 20min
tube8 tube7 tube6 tube5 tube2 tube1 Ladder
