Team:Osaka/Notebook

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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
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)

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; Amp 3μl or Kan 0.6μl 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

 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

@@ Line 1: / Line 1: @@
-{{Osaka}}
-<html>
-<div class="contents2">
-</html>
-{{Osaka/Calendar}}
 ==Notebook==
@@ Line 91: / Line 86: @@
 # 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
@@ Line 130: / Line 126: @@
 #* 1-13D, 1-2M with XbaI, PstI
 #* (RESULTS?)
-# Transformation of 2-22P, 1-2J using 25μl of competent cells each
+# 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)===
@@ Line 137: / Line 140: @@
 ===August 19 (Thu)===
-.液培へ
+# Transfer of 2-22P, 1-2J to solution culture
-    Anp 2-22P,1-2J 37℃ 10:00~
+# 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
-.電気泳動
+===August 20 (Fri)===
-    dye 2μl,サンプル 10μl,Ladder 2μl,2% Agalose gel
+# Gel electrophoresis of 1-1D and its digests
+#* (RESULTS?)
-V 30min,EtBr 30min 60min 90min
+# 'Cut check' of parts miniprepped the night before
-    Ladder,1-1D(1)(2) d,1-2M(1)(2) d,1-18F(1)(2) d,1-13D(1)(2) d
+#* both 2-22P & 1-2J cut with XbaI, PstI
+#* enzyme inactivation at 80℃, 20min
-    dye 2μl,サンプル 10μl,Ladder 2μl,1% Agalose gel
+#* (RESULTS?)
+# Restriction digest of 2-20J (WHICH ENZYMES?)
-V 30min,EtBr 30min 60min
+# Ligation according to 3A assembly method
-    Ladder,1-1D
-.制限酵素処理
-    プラスミド:1-1D
-            SpeI処理        EcoRI処理        SpeI+EcoRI処理
-    プラスミド    2.5μl            2.5μl            2.5μl
-    EcoRI        0            1μl            1μl
-    SpeI        1μl            0            1μl
-    H2O        41μl            41μl            40μl
-xNEbuffer2    5μl            5μl            5μl
-xBSA    0.5μl            0.5μl            0.5μl
-    total        50μl            50μl            50μl
-℃ incubate 20:00~
-.mini prep プラスミド回収
--22P,1-2J
-/20    中村、安本、坂
-.電気泳動
-    dye 2μl,サンプル 10μl(1-1Dのみ 2μl),Ladder 2μl,1% Agalose gel
-V 20min,EtBr 30min
-    Ladder,1-1D Spe,1-1D Eco,1-1D Spe Eco,1-1D
-.制限酵素処理
--22P,1-2J
-    プラスミド    2.5μl
-    d-H2O        40μl
-xNEbuffer    5μl
-xBSA    0.5μl
-    XbaI        1μl
-    PstI        1μl
-    total        50μl
-℃ incubate
-  電気泳動
-    dye 2μl,サンプル 10μl,Ladder 2μl,1% Agalose gel
-V 20min,EtBr 30min
-    Ladder,1-2J(1) d,1-2J(2) d,2-22P(2) d
--2J(1) d,1-2J(2) d,2-22P(2) d → 80℃,20min 失活
-. 制限酵素処理（サンプル2-20Jが見当たらなかったため）
-/9のレシピで再度制限酵素処理
    電気泳動