Team:Osaka/Notebook

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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°C, 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°C 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 001; Chloramphenicol resistance
    • same ligation mix composition as yesterday's
  3. Transformation of 001 with pre-incubation for 1.5hr instead of 1hr

August 22 (Sun)

  1. Transfer of 001 to culture solution; incubation at 30°C (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°C as per normal

August 23 (Mon)

  1. Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
  2. Miniprep & 'Cut check' of 001
    • cut at EcoRI, SpeI
    • gel run with DNA ladder, digested plasmid, undigested plasmid
    • (RESULTS?)
  3. Transfer of 3 more colonies of 001 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 001 (why?)
    • 200ml of culture solution mixed with 100ml of 50% glycerol
    • stored at -80°C

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°C 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 002
    2. 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
  5. Transformation of ligation products 002 and 003

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 002 and 003 to solution culture (3 colonies each)

August 31 (Tue)

  1. Miniprep of 002, 003
  2. 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
  3. Repeat colony pick-up and solution culture of 2-2O (5 colonies this time)

September 1 (Wed)

  1. Transformation
IDPart NameResistanceDescription
1-12D<bbpart>BBa_E2030</bbpart>Kyeast-optimized EYFP
1-12B<bbpart>BBa_E2020</bbpart>Kyeast-optimized ECFP
3-2K<bbpart>BBa_K165001</bbpart>Ayeast GAL1 promoter
1-7D<bbpart>BBa_J63006</bbpart>Ayeast GAL1 promoter + Kozak sequence
  1. Miniprep of 5 separate cultures of 2-2O inoculated yesterday
  2. 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)

  1. Colony check
    • 1-12D, 3-2K, 1-7D produced colonies -> inoculated into solution culture
    • 1-12B did not transform successfully
  2. 3A assembly ligations:
    1. 001 as upstream, 1-2J as downstream, 1-3A as vector; product designated as 004
    2. 001 as upstream, 2-22P as downstream, 1-3A as vector; product designated as 005
  3. Transformation of ligation products

September 3 (Fri)

  1. 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)
  2. Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI
    • all lengths ok
  3. Transfer of yesterday's transformations (colonies appeared later in the evening) to culture solution (2 colonies picked up from each plate)

September 4 (Sat)

  1. Miniprep of 004, 005
  2. Restriction digest of 004, 005 and 1-7D (as control) with EcoRI, SpeI
  3. 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 -> ??
  4. Gel electrophoresis followed by purification of 001 to isolate insert -> Standard Assembly
    • gel purification performed according to protocol in QIAquick Spin Handbook
  5. 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)

  1. Heat inactivation of yesterday's ligation mixes at 80°C for 20min followed by transformation.

September 6 (Mon)

  1. Transfer of yesterday's transformations to solution culture
  2. Transformation of the following registry parts:
IDPart NameResistanceDescription
2-10F<bbpart>BBa_K081005</bbpart>Aconstitutive promoter + RBS
2-10H<bbpart>BBa_K081006</bbpart>Alambda phage promoter + RBS

September 7 (Tue)

  1. Miniprep of 004, 005
  2. Cut check with EcoRI, SpeI
    • both insert lengths ok!
  3. Transformation of DNA for PGA synthesis-related genes
IDPart NameResistanceDescription
A01pTPG01-1Aplasmid pTrc99A with pgs genes inserted
A02pTPG01-2A''
A03pBSGR3Kglutamine racemase
  1. 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)

  1. Miniprep 2-10F, 2-10H, 004, 005

２．制限酵素処理 

   2-10F,2-10H,004,005

　　　　　　　　　　　　　　　　　　　　　ネガティブコントロール プラスミド 2.5μｌ 　　　　　 2.5μｌ EcoRI 　　　　　0.5μｌ　 　　　　　0.5μｌ Spe I　　　　　　　　0.5μｌ　 　　　　　　　0μｌ 10×NEbuffer2　　　5μｌ 　 　　　　 　 5μｌ 100×BSA　　　　　　0.5μｌ 　　　　　 0.5μｌ 　H2O　　　　　　　 41μｌ 　　　　　 41.5μｌ total 50μｌ　　　　　　　　　　　　 50μｌ　　　　　　

37℃ incubate 30min

３．電気泳動 　　dye 2μｌ、サンプル　10μｌ、Ladder　2μｌ 

 1%Agalose gel、100V　20min, EtBr 1h10min
    EtBrは新しいものにかえた。10μg/μl　30μlを300mlに

005 005 004 004 2-10H 2-10H 2-10F 2-10F Ladder 

E      ES    E      ES      E          ES       E          ES

４．PCR 　　　　　　　　　　mix組成 

              Ex taq                  0.25μｌ
             10×taq Buffer          5μｌ
              dNTP  Mix              4μｌ
               Template             1μｌ
               primer  rev           0.5μｌ
                            fwr           0.5μｌ
                  DSW                 38.75μｌ
                 total                     50μｌ

  94℃  2min

　　３０サイクル 

  94℃   30sec                         tube6     cenA
  68℃   30sec                          tube7     cex
  72℃   2min                            tube8      bgl

５．電気泳動 

 　dye 2μｌ、サンプル　5μｌ、Ladder　2μｌ
 1%Agalose gel、100V　, EtBr
  Ladder, 6 ,7 ,8             PCR後そのまま

６．電気泳動 　　　dye 2μｌ、サンプル　10μｌ、Ladder　2μｌ 

1%Agalose gel、100V　   (PCR purification Ladder)

左から8(プラスミド)7(プラスミド)6(プラスミド) 8(PCR)7(PCR)6(PCR)Ladder

PCRができていない。 Templateのみがバンドで見られる。 

8の8kbpのバンドは何？

７．再度PCR 

               Ex taq                  0.25μｌ
             10×taq Buffer          5μｌ
              dNTP  Mix              4μｌ
               Template             1μｌ
               primer  rev           0.5μｌ
                            fwr           0.5μｌ
                  DSW                 38.75μｌ
                 total                     50μｌ

    tube    1: F1×100
               2: F1×1000
               3: 2-20H×100
               4: 2-20H×1000
               5: 2-20J×100
               6: 2-20J×1000

８． A01,A02,A03 を液培へ 

  左２つにAmp 3μｌ      残りにK 0.6μｌ

→37℃ incubate 23:10~

９．電気泳動 

 5×dye 1μｌ、サンプル　5μｌ、　マーカー2μｌ
 1%Agalose gel、100V　25min, EtBr 20min

　　PCR産物が見られない。アニーリング温度が原因か？

１０．再再度PCR 

                　　　　mix組成                 
              Ex taq                  0.25μｌ
             10×taq Buffer          5μｌ
              dNTP  Mix              4μｌ
               Template             1μｌ
               primer  rev           0.5μｌ
                            fwr           0.5μｌ
                  DSW                 38.75μｌ
                 total                     50μｌ

　　　　サンプル番号 

  1. F1×10                                  94℃  1min
  2. 2-20H×10      →                   98℃  10sec
  3. 2-20J×10                              65℃  30sec       下３つを30cycle
                                        　         72℃ 2min  

  4. F1×10                                    94℃  2min
  5.2-20H×10         →                   98℃  10sec       30cycle
  6.2-20J×10                                68℃  2min

　４．プライマー異なるものも入ってしまった　　テンプレート入れすぎ

１１．再々度PCR　電気泳動 　　　以下９と同じ 左からLadder Sample 1 2 3 4 5 6

Annealing temp 低いか？ ※ 今後PCRの条件をどう設定していくか？

ゲルの結果 

   Sample1:16721bp(これは正解)
   Sample2:これは失敗
   Sample3:1350bp付近にバンドあり。目的遺伝子は増幅できた。
   Sample4:
   Sample5:1461bp付近にバンドあり。目的遺伝子は増幅できた。
   Sample6:1353bp付近にバンドあり。目的遺伝子は増幅できた。

 Sample2を除き　45μl電気泳動     Gel purification を行うことにした。
 この時点でF1はprimerの設計ミスにより、Assemblyの対象から

　外し、破棄した。(Sample1,Sample4　破棄)

１２．Gel 　purification 　　再々度PCR Sample 3, 5 ,6 ,8を　電気泳動　　Gel purification 

    ladder     4μｌ
    loading    41


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