Team:Osaka/Notebook

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#* 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates)
#* 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~　

Revision as of 13:00, 6 October 2010

Calendar

July
S M T W T F S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
August
S M T W T F S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
September
S M T W T F S
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
October
S M T W T F S
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
November
S M T W T F S
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30











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:
IDPart NameResistanceDescription
2-20J<bbpart>BBa_K118023</bbpart>AC. fermi endocellulase Cen A coding
2-20H<bbpart>BBa_K118022</bbpart>AC. fermi exocellulase Cex coding
1-2M<bbpart>BBa_B0034</bbpart>ARBS
1-13D<bbpart>BBa_B0010</bbpart>Aterminator
1-1D<bbpart>BBa_R0010</bbpart>Apromoter
1-18F<bbpart>BBa_E1010</bbpart>KRFP 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
IDPart NameResistanceDescription
1-1C<bbpart>pSB1A3</bbpart>Aconstruction 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; 3μl Amp or 0.6μl Kan solution 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
IDPart NameResistanceDescription
2-22P<bbpart>BBa_K103006</bbpart>AOmpA outer membrane protein + linker
1-2J<bbpart>BBa_J32015</bbpart>A,KPelB 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: 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
  5. 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)

  1. 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
  2. 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
  3. Transformation of <1> with pre-incubation for 1.5hr instead of 1hr

August 22 (Sun)

  1. Transfer of <1> to culture solution; incubation at 30℃ (why??)
  2. Transformation of the following parts:
IDPart NameResistanceDescription
2-4A<bbpart>BBa_J63005</bbpart>Ayeast ADH1 promoter
F1N/AAbeta-galactosidase from Edinburgh team
F2N/ACRBS + F1
F3N/ACLac promoter + RBS + F1
    • O/N incubation at 37℃ as per normal

August 23 (Mon)

  1. Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
  2. Miniprep & 'Cut check' of <1>
    • cut at EcoRI, SpeI
    • gel run with DNA ladder, digested plasmid, undigested plasmid
    • (RESULTS?)
  3. Transfer of 3 more colonies of <1> to liquid solution (to store as glycerol stock - see Tue notes)
  4. Transformation of the following registry parts
IDPart NameResistanceDescription
2-2O<bbpart>J63003</bbpart>Ayeast Kozak sequence
3-11I<bbpart>K105027</bbpart>A'cyc100' minimal promoter

August 24 (Tue)

  1. Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
    • transfer to solution culture
  2. Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
    • (RESULTS)
  3. Transfer of F1 to solution culture (why?)
  4. 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)

  1. Miniprep of parts in solution culture: 2-2O, 3-11I, F1
  2. Cut check of 3-11I & F1 with EcoRI, SpeI
    • (RESULTS?)

August 26 (Thu)

  1. 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)

  1. Transfer of yesterday's transformed parts (all produced colonies) to solution culture
  2. Transformation of the following parts
IDPart NameResistanceDescription
1-1K<bbpart>BBa_J63010</bbpart>AProtein fusion vector (Silver standard)
1-1I<bbpart>BBa_J63009</bbpart>ALow copy protein fusion vector (Silver standard)
3-3G<bbpart>BBa_K157013</bbpart>A15aa glycine-serine linker (Freiburg standard)
    • using competent cells opened on 8/20
  2. Preparation of YPD yeast culture medium with the following recipe:
MiliQ water1 liter
Bacto tryptone20.0g2%
Bacto yeast extract10.0g1%
Glucose20.0g2%
    • pH was adjusted to 5.8
    • autoclaved before use
    • 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
  2. Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar

August 28 (Sat)

  1. Miniprep of parts in solution culture
  2. 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
  3. Gel electrophoresis of digests
    • Plasmids not detected for 2-4A & 3-11I - mistake during miniprep? culture duration too long, plasmid loss occurred?
  4. 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)

  1. Miniprep: 1-1K, 1-1I, 3-3G, 2-4A, 3-11I
  2. Restriction digests
    • for checking: 1-1K, 1-1I, 3-3G with EcoRI, PstI
    • for assembly: 2-4A, 3-11I with EcoRI, SpeI (upstream parts)
  3. Gel electrophoresis for confirmation
    • Inserts seem to be present in all samples
  4. 3A assembly ligations:
    1. 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as <2>
    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℃
  5. Transformation of ligation products <2> and <3>

August 30 (Mon)

  1. 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
  2. Gel electrophoresis of the digests to confirm inserts
    • all OK
  3. 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~　


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