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From 2010.igem.org

Contents 1 Notebook 1.1 July 29 (Thu) 1.2 July 31 (Sat) 1.3 August 2 (Mon) 1.4 August 3 (Tue) 1.5 August 4 (Wed) 1.6 August 5 (Thu) 1.7 August 6 (Fri) 1.8 August 7 (Sat) 1.9 August 8 (Sun) 1.10 August 9 (Mon) 1.11 August 10 (Tue) 1.12 August 11 (Wed) 1.13 August 16 (Mon) 1.14 August 17 (Tue) 1.15 August 18 (Wed) 1.16 August 19 (Thu) 1.17 August 20 (Fri)

Notebook

July 29 (Thu)

Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura

1. Safety lecture for junior members.
2. Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).

July 31 (Sat)

Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka

1. Meeting
   • Summer project schedule
   • List of genes to clone

August 2 (Mon)

Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka

1. Culture medium preparation
   • LB agar plates (49 antibiotic-less plates)
   • LB liquid medium (500 ml)
2. 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

1. 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

1. OD measurements throughout the day till required OD (0.3~0.7) was obtained.
2. Completion of competent cells according to protocol.

August 5 (Thu)

Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino

1. 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

1. 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
2. Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C

August 7 (Sat)

Attendance: Nakamura, Saka, Yasumoto, Takino

1. Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
2. 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	(" ")

1. Meeting

August 8 (Sun)

Attendance: Nakamura, Yasumoto

1. Colony check
   • All parts successfully transformed
2. Transfer to LB culture medium

August 9 (Mon)

1. Miniprep of 1-1C
2. Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
   • (WHICH ENZYMES?)
3. 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)
4. Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
   • Yesterday's inoculated culture mediums contained the wrong antibiotics!
5. 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)

1. Miniprep of 1-3A, 1-5A
2. Restriction digests of 1-3A, 1-5A
3. Gel electrophoresis
   1. 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)
   2. 2nd run: 1-1D, 1-18F, 1-13D, 1-2M (2nd repeat)
      • all parts not cut

August 11 (Wed)

1. Miniprep of last year's parts transformed on Monday
2. 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
3. Sent miniprepped last year's parts to ECUST team in Shanghai, China

August 16 (Mon)

1. 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
2. Gel electrophoresis of digests
   • (RESULTS?)
3. Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D,　1-2M,　1-1D,　1-18F
   • 3ml LB liquid medium; Amp 3μl or Kan 0.6μl added

August 17 (Tue)

1. Mini-prep of 1-13D,　1-2M,　1-1D,　1-18F parts inoculated yesterday (2 of each)
2. '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?)
3. 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)

1. Transfer of 2-22P, 1-2J to solution culture
2. 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?)
3. Gel electrophoresis of 1-1D digest only
   • (RESULT?)
4. Multiple restriction digests of 1-1D to check for problems at restriction sites
   • tried the following: EcoRI only; SpeI only; EcoRI + SpeI
5. Night: miniprep of 2-22P, 1-2J inoculated in the morning

August 20 (Fri)

1. Gel electrophoresis of 1-1D and its digests
   • (RESULTS?)
2. 'Cut check' of parts miniprepped the night before
   • both 2-22P & 1-2J cut with XbaI, PstI
   • enzyme inactivation at 80℃, 20min
   • (RESULTS?)
3. Restriction digest of 2-20J (WHICH ENZYMES?)
4. Ligation according to 3A assembly method

 電気泳動
   100V 20min,EtBr
   2-20J d,Ladder
   2-20J → 80℃,20min 失活

4.ライゲーション 3A assembly

   制限処理したプラスミドを使用
   2-20J                2μl    2-20J    　2μl
   2-22P                2μl    1-2J(1)    　2μl
   1-3A                2μl        　2μl
   10xT4 DNA Ligasebuffer    2μl        　2μl
   T4 DNA Ligase        1μl        　1μl
   dH2O                11μl        　11μl
   total                20μl        　20μl

   2-20H                2μl    2-20H    　2μl
   2-22P                2μl    1-2J(1)    　2μl
   1-3A                2μl        　2μl
   10xT4 DNA Ligasebuffer    2μl        　2μl
   T4 DNA Ligase        1μl        　1μl
   dH2O                11μl        　11μl
   total                20μl        　20μl

   room temprature 10min
   80℃,20min 失活
  

5.トランスフォーメーション

   8/17と同じ手順
   コンピ(2) 50μl使用  DNA 2μl
   クォーラロフェニコールPlateに150μlをまく
   37℃ incubate 16:10~

8/21 中村、安本、坂 1.液培へ

   2-20J 2-22P(1)(2),2-20J 1-2J(1)(2)(3),2-20H 2-22P(1)(2),2-20H 1-2J(1)(2)(3)
   Chr 1μl,LB 3ml
   30℃ incubate 13:30~

2.ライゲーション

   8/20と同じレシピ
   1-1D,1-2M,1-3Aをライゲーション

3.トランスフォーメーション

   SOC 10μl
   37℃ 1h → 1.5h
   Plate(c)にまく 16:40~ 37℃ incubate