Team:Osaka/Notebook

From 2010.igem.org

(Difference between revisions)
Line 573: Line 573:
# Restriction digests for 3A assembly
# Restriction digests for 3A assembly
#* 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
#* 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
-
#* 2-20J (CenA), 2-20H (Cex), F1 (BglX) with XbaI, PstI
+
#* 2-20J (CenA), 2-20H (Cex), F1 with XbaI, PstI
-
#** ''beta-glucosidase received from Edinburgh team temporarily designated BglX''
+
# Gel electrophoresis of the digests to confirm inserts
# Gel electrophoresis of the digests to confirm inserts
#* all OK
#* all OK
Line 640: Line 639:
!ID!!Part Name!!Resistance!!Description
!ID!!Part Name!!Resistance!!Description
|-
|-
-
|2-10F||<bbpart>BBa_K081005</bbpart>||A||constitutive promoter + RBS
+
|2-10F||<bbpart>BBa_K081005</bbpart>||A||constitutive promoter from combinatorial library + RBS
|-
|-
|2-10H||<bbpart>BBa_K081006</bbpart>||A||lambda phage promoter + RBS
|2-10H||<bbpart>BBa_K081006</bbpart>||A||lambda phage promoter + RBS
Line 670: Line 669:
#* ''new batch of EtBr for staining''
#* ''new batch of EtBr for staining''
#* (RESULTS?)
#* (RESULTS?)
-
# Tranfer of A01~A03 to solution culture
+
# Transfer of A01~A03 to solution culture
-
# PCR to make Silver standard-compatible parts from 2-20J (CenA), 2-20J (Cex), F1 (BglX) based on protocol in Takara Ex Taq polymerase kit
+
# PCR to make Silver standard-compatible parts from 2-20J (CenA) and 2-20J (Cex) based on protocol in Takara EX Taq polymerase kit
#* it took several tries to get a successful reaction
#* it took several tries to get a successful reaction
#** 1st attempt: template DNA was used directly; concentration too high (failure)?
#** 1st attempt: template DNA was used directly; concentration too high (failure)?
Line 681: Line 680:
===September 9 (Thu)===
===September 9 (Thu)===
# Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
# Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
-
# Purification of PCR product from gel using QIA Quick Spin gel extraction kit (''why not use PCR purification kit??'')
+
# Purification of 9/8 PCR products from gel using QIA Quick Spin gel extraction kit (''why not use PCR purification kit??'')
-
# Restriction digest of PCR products with XbaI, PstI
+
# Restriction digest of A01~03 with EcoRI, PCR products with XbaI, PstI
-
# Gel electrophoresis of digested A01, A02, A03, FcenA, Fcex, 1-5A <- ''when was this digested with X, P?''
+
# Gel electrophoresis of digested parts together with 1-5A ''1-5A supposed to be receiving vector, but digested at wrong sites''
-
# Another round of PCR to amplify Cex
+
#* CenA PCR product -> OK (Silver-compatible part designated FcenA)
 +
#* Cex PCR product -> ?
 +
# Another round of PCR to amplify Cex as Silver standard part (''why?'')
#* 10X dilution of template
#* 10X dilution of template
#* 68°C annealing temp
#* 68°C annealing temp
Line 691: Line 692:
#* 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
#* 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
#* 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
#* 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
-
#* 008: 3A assembly of 001 (upstream), Fcex (downstream), 1-5A (vector)
+
#* 008: 3A assembly of 001 (upstream), Cex PCR product (downstream), 1-5A (vector) ''bad insert?''
#* 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
#* 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
# Transformation of newly assembled parts 006~009
# Transformation of newly assembled parts 006~009
Line 697: Line 698:
===September 10 (Fri)===
===September 10 (Fri)===
-
# Gel electrophoresis of amplified <i>cex</i>, and 2-20H.
+
# Gel electrophoresis of PCR product from Cex and 2-20H (original Cex part) for comparison
-
# Gel purification of amplified <i>cex</i>.
+
#* PCR product seems ok -> purification from gel; Silver-compatible part designated Fcex
-
# Restriction digest Fcex plasmid, FcenA plasmid with　<i>Xba</i>I, <i>Pst</i>I and 004, 005 with <i>Eco</i>RI, <i>Spe</i>I.
+
# Restriction digests of 004 & 005 (9/4 ligations) with EcoRI, SpeI; followed by gel electrophoresis
-
# Gel electrophoresis of digested 004, 005 and Fcex, FcenA, PCR product(FcenA).
+
#* (RESULTS?)
-
# The result of transformation in 9/9
+
# Restriction digests of Fcex (purified today), FcenA (amplified on 9/9) with　XbaI, PstI followed by gel electrophoresis
-
#* 008 more than 100 colonies
+
#* (RESULTS?)
-
#* 009 more than 100 colonies
+
# Ligations
-
#* FcenA more than 100 colonies
+
#* Fcex: PCR product from Cex with 1-5A as vector (cut/ligated at X, P)
-
#* 006 none
+
#* 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A　(vector); ''repeat''
-
#* 007 none
+
#* 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector); ''repeat''
-
# Ligation
+
-
#* 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A　(vector)  
+
-
#* 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)  
+
-
#* Fcex: Fcex with 1-5A as vector (cut/ligated at X, P)
+
#* 010: 3A assembly of 004 (upstream), Fcex (downstream), 1-5A (vector)  
#* 010: 3A assembly of 004 (upstream), Fcex (downstream), 1-5A (vector)  
#* 011: 3A assembly of 005 (upstream), Fcex (downstream), 1-5A (vector)
#* 011: 3A assembly of 005 (upstream), Fcex (downstream), 1-5A (vector)
-
# Transformation of newly assembled parts 006, 007, Fcex, 010, 011.
+
# Transformation of newly assembled parts Fcex, 006, 007, 010, 011.
-
# Transfer of 008, 009, FcenA, 001, 2-10F to solution culture.
+
# Colony check of 9/9 transformations
 +
#* 006: no colonies
 +
#* 007: no colonies
 +
#* 008: >100 colonies ''bad insert?''
 +
#* 009: >100 colonies
 +
#* FcenA: >100 colonies
 +
# Transfer of 008, 009, FcenA, 001, 2-10F to solution culture. (''more 001, 2-10F needed'')
===September 11 (Sat)===
===September 11 (Sat)===
# Miniprep of 008, 009, FcenA, 001, 2-10F.
# Miniprep of 008, 009, FcenA, 001, 2-10F.
-
# Ristriction digest of 008, 009, 001, 2-10F with <i>Eco</i>RI,　<i>Spe</i>I and FcenA with　<i>Xba</i>I, <i>Pst</i>I.
+
# Ristriction digest of 008, 009, 001, 2-10F with EcoRI,　SpeI and FcenA with XbaI, PstI.
# Gel electrophoresis of digested  008, 009, FcenA, 001, 2-10F.
# Gel electrophoresis of digested  008, 009, FcenA, 001, 2-10F.
-
#* 009 -> O.K.
 
#* 008 -> ???
#* 008 -> ???
-
#* Add 1-13D as terminator to 008 and 009.
+
#* 009 -> O.K.
-
#* FcenA was not digested by <i>Xba</i>I.
+
#* ''add 1-13D as terminator to 008 and 009'
-
# Restriction digest of FcenA with <i>Eco</i>RI.
+
#* ''FcenA was not digested by XbaI''
-
# Ligation
+
# Restriction digest of FcenA with EcoRI.
-
#* 012: 3A assembly of 009 (upstream), 1-13D(terminator,　downstream), 1-3A (vector)
+
# Gel electrophoresis of FcenA
-
# Gel electrophoresis of FcenA.
+
#* FcenA was digested by EcoRI -> O.K.
-
#* FcenA was digested by <i>Eco</i>RI. -> O.K.
+
# Ligations for 3A assembly
-
# Ligation
+
#* 012: 009 (upstream), 1-13D(terminator,　downstream), 1-3A (vector)
-
#* 013: 3A assembly of 008 (upstream), 1-13D(terminator,　downstream), 1-5A (vector)
+
#* 013: 008 (upstream), 1-13D(terminator,　downstream), 1-5A (vector) ''bad insert?''
-
# Transfomation of newly assembled parts 012, 013.
+
# Transfomation of newly assembled parts 012, 013
-
# Transfer of 012, 013 to solution culture.
+
# Transfer of Fcex, 006, 007, 010, 011 (transformed yesterday) to solution culture.
 +
 
 +
===September 12 (Sun)===
 +
# Miniprep of Fcex, 006, 007, 010, 011
 +
# Restriction digests of 006, 007, 010, 011 with EcoRI, SpeI; Fcex with XbaI, PstI
 +
# Gel electrophoresis of digests
 +
#* (RESULTS)
 +
# Ligation for 3A assembly
 +
#* 014: 011 as upstream, 1-13D as downstream, 1-3A as vector
 +
# Transformation of 014 using 2μl ligation product with 50μl competent cells
 +
# Moved yesterday's transformation plates to 4°C refrigerator ''no RFP expression from vector plasmids at all?''
 +
# Transfer of A01, A02, A03 (previously transformed) to solution culture
 +
 
 +
===September 13 (Mon)===
 +
# Transfer to culture solution: 011, 012 ''RFP expressed during refrigeration; selection became possible''
 +
# Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis
 +
 
 +
(''beta-glucosidase received from Edinburgh team temporarily designated BglX'')
 +
 
 +
5.PCR  beta-guluctosidase
 +
  thermal cycle
 +
          94℃      2min
 +
          98℃      10sec
 +
          68℃      2min
 +
 
 +
　　　　　　　　　　mix組成               
 +
              Ex taq                    0.25μｌ
 +
            10×taq Buffer            5μｌ
 +
              dNTP  Mix                4μｌ
 +
              Template(×10)        1μｌ
 +
              primer  beta-gal      0.5μｌ
 +
                          malz rev      0.5μｌ
 +
                  DSW                  38.75μｌ
 +
                total                      50μｌ
 +
　　　プライマーが間違ってるがプラスミド存在確認のためにやる。
 +
 
 +
    pgsC      451bp
 +
      thermal cycle
 +
            94℃      2min 
 +
            98℃    10sec
 +
            55℃      30sec
 +
            72℃      40sec
 +
 
 +
　　　　　　　　　mix組成               
 +
              Ex taq                            0.25μｌ
 +
            10×taq Buffer                  5μｌ
 +
              dNTP  Mix            　　      4μｌ
 +
              Template(×10)(×100)    1μｌ
 +
              primer  rev                  0.5μｌ
 +
                            fwd                  0.5μｌ
 +
                  DSW                      38.75μｌ
 +
                total                            50μｌ
 +
 
 +
６．電気泳動
 +
A01,A03  5μｌ
 +
A01d, A03d　40μｌ
 +
dye 2μｌ、Ladder　2μｌ
 +
1%Agalose gel、100V　20min  EtBr 30min
 +
 
 +
A01  ,A01d  ,A03  ,A03d  ,Ladder
 +
 
 +
７．ＰＣＲ産物電気泳動
 +
PCRdye 1μｌ、サンプル　5μｌ、Ladder　2μｌ
 +
1%Agalose gel、100V　20min  EtBr
 +
      2          1          Ladder
 +
 
 +
8.Gel purification      beta-guluctosidase    ＰＣＲ産物
 +
dye 4μｌ、サンプル　45μｌ、Ladder　4μｌ
 +
1%Agalose gel、100V　20min  EtBr
 +
 
 +
９．液培へ（9月13日①②の分が赤かったため）
 +
　　　012③、④
 +
　　　013③、④
 +
　　　　37℃  incubate  22:15~
 +
 
 +
 
 +
１０．制限酵素処理
 +
    β-Gluctosidase(PCR→gel purification), 1-5A
 +
　DNA(G-G)            2.5μｌ
 +
　EcoRI    　　　　　0.5μｌ　
 +
SpeI                　　 0.5μｌ　
 +
10×NEbuffer2　　　5μｌ
 +
100×BSA　　　　　　0.5μｌ
 +
　H2O　　　　　　　  38.5μｌ
 +
total                      50μｌ　
 +
 
 +
1-5A                      2.5μｌ
 +
　EcoRI    　　　　　0.5μｌ　
 +
SpeI                　　 0.5μｌ　
 +
10×NEbuffer2　　　5μｌ
 +
100×BSA　　　　　　0.5μｌ
 +
　H2O　　　　　　　  38.5μｌ
 +
total                      50μｌ　
 +
 
 +
１１．ＰＣＲ産物（PgsC（仮））電気泳動
 +
　dye 1μｌ、サンプル　5μｌ、マーカー2μｌ
 +
４００－５００bpのあｔりのバンドが見られた。ＰＣＲ条件がわかった！
 +
 
 +
１２．A01  A02  A03  トランスフォーメーション
 +
　　　コンピ②50μｌ＋ＤＮＡ2μｌ
 +
　　　　プレートにまく
 +
 
 +
１３．014を液培（LB medium,C）に移す  ①、②、③
 +
 

Revision as of 04:53, 11 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 (See Table 1).

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.

Table 1
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

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 (See Table 2)
  3. Meeting
Table 2
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(" ")

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(See Table 3)
Table 3
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 (See Table 4)
    • O/N incubation at 37°C as per normal
Table 4
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

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 (See Table5)
Table 5
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 (See Table 6)
    • using competent cells opened on 8/20
  3. Preparation of YPD yeast culture medium with the following recipe (See Table 7)
    • pH was adjusted to 5.8
    • autoclaved before use
    • 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
  4. Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar
Table 6
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)
Table 7
MiliQ water1 liter
Bacto tryptone20.0g2%
Bacto yeast extract10.0g1%
Glucose20.0g2%

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 with XbaI, PstI
  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 (See Table 7)
  2. Miniprep of 5 separate cultures of 2-2O inoculated yesterday
  3. 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?
Table 7
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

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 (See Table 8)
Table 8
IDPart NameResistanceDescription
2-10F<bbpart>BBa_K081005</bbpart>Aconstitutive promoter from combinatorial library + 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 (See Table 9)
  4. Transfer to solution culture
    • 004, 005 transformed on 9/5 (pick up from fresh colonies) -> needed more plasmid
    • 2-10F, 2-10H transformed yesterday
Table 9
IDPart NameResistanceDescription
A01pTPG01-1Aplasmid pTrc99A with pgs genes inserted
A02pTPG01-2A''
A03pBSGR3Kglutamine racemase

September 8 (Wed)

  1. Miniprep 2-10F, 2-10H, 004, 005
  2. Restriction digest of above parts with EcoRI, SpeI; also with only EcoRI as negative control
  3. Gel electrophoresis
    • new batch of EtBr for staining
    • (RESULTS?)
  4. Transfer of A01~A03 to solution culture
  5. PCR to make Silver standard-compatible parts from 2-20J (CenA) and 2-20J (Cex) based on protocol in Takara EX Taq polymerase kit
    • it took several tries to get a successful reaction
      • 1st attempt: template DNA was used directly; concentration too high (failure)?
      • 2nd attempt: 100X, 1000X dilutions attempted without success; this time, over-dilution or stringent annealing temp (68°C) may have been culprit?
      • 3rd attempt: 10X dilutions, annealing temp lowered to 65°C -> success!
    • note: reactions were evaluated by gel electrophoresis of crude PCR product - if band appears at approximately correct length then reaction judged as successful
    • special note of thanks to Nakamura who stayed in lab overnight to run the PCRs

September 9 (Thu)

  1. Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
  2. Purification of 9/8 PCR products from gel using QIA Quick Spin gel extraction kit (why not use PCR purification kit??)
  3. Restriction digest of A01~03 with EcoRI, PCR products with XbaI, PstI
  4. Gel electrophoresis of digested parts together with 1-5A 1-5A supposed to be receiving vector, but digested at wrong sites
    • CenA PCR product -> OK (Silver-compatible part designated FcenA)
    • Cex PCR product -> ?
  5. Another round of PCR to amplify Cex as Silver standard part (why?)
    • 10X dilution of template
    • 68°C annealing temp
  6. Ligation
    • FcenA: PCR product with 1-5A as vector (cut/ligated at X, P)
    • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
    • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
    • 008: 3A assembly of 001 (upstream), Cex PCR product (downstream), 1-5A (vector) bad insert?
    • 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
  7. Transformation of newly assembled parts 006~009
  8. Transfer of 006~009 to solution culture.

September 10 (Fri)

  1. Gel electrophoresis of PCR product from Cex and 2-20H (original Cex part) for comparison
    • PCR product seems ok -> purification from gel; Silver-compatible part designated Fcex
  2. Restriction digests of 004 & 005 (9/4 ligations) with EcoRI, SpeI; followed by gel electrophoresis
    • (RESULTS?)
  3. Restriction digests of Fcex (purified today), FcenA (amplified on 9/9) with　XbaI, PstI followed by gel electrophoresis
    • (RESULTS?)
  4. Ligations
    • Fcex: PCR product from Cex with 1-5A as vector (cut/ligated at X, P)
    • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A　(vector); repeat
    • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector); repeat
    • 010: 3A assembly of 004 (upstream), Fcex (downstream), 1-5A (vector)
    • 011: 3A assembly of 005 (upstream), Fcex (downstream), 1-5A (vector)
  5. Transformation of newly assembled parts Fcex, 006, 007, 010, 011.
  6. Colony check of 9/9 transformations
    • 006: no colonies
    • 007: no colonies
    • 008: >100 colonies bad insert?
    • 009: >100 colonies
    • FcenA: >100 colonies
  7. Transfer of 008, 009, FcenA, 001, 2-10F to solution culture. (more 001, 2-10F needed)

September 11 (Sat)

  1. Miniprep of 008, 009, FcenA, 001, 2-10F.
  2. Ristriction digest of 008, 009, 001, 2-10F with EcoRI,　SpeI and FcenA with XbaI, PstI.
  3. Gel electrophoresis of digested 008, 009, FcenA, 001, 2-10F.
    • 008 -> ???
    • 009 -> O.K.
    • add 1-13D as terminator to 008 and 009'
    • FcenA was not digested by XbaI
  4. Restriction digest of FcenA with EcoRI.
  5. Gel electrophoresis of FcenA
    • FcenA was digested by EcoRI -> O.K.
  6. Ligations for 3A assembly
    • 012: 009 (upstream), 1-13D(terminator,　downstream), 1-3A (vector)
    • 013: 008 (upstream), 1-13D(terminator,　downstream), 1-5A (vector) bad insert?
  7. Transfomation of newly assembled parts 012, 013
  8. Transfer of Fcex, 006, 007, 010, 011 (transformed yesterday) to solution culture.

September 12 (Sun)

  1. Miniprep of Fcex, 006, 007, 010, 011
  2. Restriction digests of 006, 007, 010, 011 with EcoRI, SpeI; Fcex with XbaI, PstI
  3. Gel electrophoresis of digests
    • (RESULTS)
  4. Ligation for 3A assembly
    • 014: 011 as upstream, 1-13D as downstream, 1-3A as vector
  5. Transformation of 014 using 2μl ligation product with 50μl competent cells
  6. Moved yesterday's transformation plates to 4°C refrigerator no RFP expression from vector plasmids at all?
  7. Transfer of A01, A02, A03 (previously transformed) to solution culture

September 13 (Mon)

  1. Transfer to culture solution: 011, 012 RFP expressed during refrigeration; selection became possible
  2. Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis

(beta-glucosidase received from Edinburgh team temporarily designated BglX)

5.PCR beta-guluctosidase 

  thermal cycle
          94℃      2min
          98℃       10sec
          68℃       2min

　　　　　　　　　　mix組成                 
             Ex taq                    0.25μｌ
            10×taq Buffer            5μｌ
             dNTP  Mix                4μｌ
              Template(×10)        1μｌ
              primer  beta-gal      0.5μｌ
                          malz rev      0.5μｌ
                 DSW                   38.75μｌ
                total                       50μｌ

　　　プライマーが間違ってるがプラスミド存在確認のためにやる。 

    pgsC      451bp
     thermal cycle
            94℃       2min  
            98℃     10sec
            55℃      30sec
            72℃       40sec

　　　　　　　　　mix組成 

             Ex taq                            0.25μｌ
            10×taq Buffer                   5μｌ
             dNTP  Mix            　　       4μｌ
              Template(×10)(×100)    1μｌ
              primer  rev                   0.5μｌ
                           fwd                  0.5μｌ
                 DSW                       38.75μｌ
                total                            50μｌ

６．電気泳動 A01,A03 5μｌ A01d, A03d　40μｌ dye 2μｌ、Ladder　2μｌ 1%Agalose gel、100V　20min EtBr 30min

A01 ,A01d ,A03 ,A03d ,Ladder

７．ＰＣＲ産物電気泳動 PCRdye 1μｌ、サンプル　5μｌ、Ladder　2μｌ 1%Agalose gel、100V　20min EtBr 

      2          1           Ladder

8.Gel purification beta-guluctosidase ＰＣＲ産物 dye 4μｌ、サンプル　45μｌ、Ladder　4μｌ 1%Agalose gel、100V　20min EtBr

９．液培へ（9月13日①②の分が赤かったため） 　　　012③、④ 　　　013③、④ 　　　　37℃ incubate 22:15~


１０．制限酵素処理 

    β-Gluctosidase(PCR→gel purification), 1-5A

　DNA(G-G) 2.5μｌ 　EcoRI 　　　　　0.5μｌ　 

SpeI                 　　 0.5μｌ　

10×NEbuffer2　　　5μｌ 100×BSA　　　　　　0.5μｌ 　H2O　　　　　　　 38.5μｌ 

total                      50μｌ　

1-5A                      2.5μｌ

　EcoRI 　　　　　0.5μｌ　 

SpeI                 　　 0.5μｌ　

10×NEbuffer2　　　5μｌ 100×BSA　　　　　　0.5μｌ 　H2O　　　　　　　 38.5μｌ 

total                      50μｌ　

１１．ＰＣＲ産物（PgsC（仮））電気泳動 　dye 1μｌ、サンプル　5μｌ、マーカー2μｌ ４００－５００bpのあｔりのバンドが見られた。ＰＣＲ条件がわかった！

１２．A01 A02 A03 トランスフォーメーション 　　　コンピ②50μｌ＋ＤＮＡ2μｌ 　　　　プレートにまく

１３．014を液培（LB medium,C）に移す ①、②、③


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