Team:Osaka/Notebook

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Revision as of 16:35, 6 October 2010

Calendar

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:

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

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

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:

1. • 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

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

1. • using competent cells opened on 8/20
2. Preparation of YPD yeast culture medium with the following recipe:

1. • 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 of <2>, <3>
2. Cut check of <2>, <3> with EcoRI, SpeI
   • <3> was properly cut, but the insert length was inconsistent; looking back at 8/28 gel result, length of 2-2O (downstream part in <3>) 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

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. <1> as upstream, 1-2J as downstream, 1-3A as vector; product designated as <4>
   2. <1> as upstream, 2-22P as downstream, 1-3A as vector; product designated as <5>
3. Transformation of ligation products

September 3 (Fri)

1. Colony check: yesterday's transformations seem to have failed; repeat of transformations of <4> and <5> 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 <4>, <5>
2. Restriction digest of <4>, <5> and 1-7D (as control) with EcoRI, SpeI
3. Gel electrophoresis
   • 1-7D -> OK
   • <4> -> insert length same as <1>; since both upstream part <1> and vector 1-3A were C resistance, 3A assembly must have failed to yield ligation product; try Standard Assembly!
   • <5> -> ??
4. Gel electrophoresis followed by purification of <1> to isolate insert -> Standard Assembly
   • gel purification performed according to protocol in QIAquick Spin Handbook
5. Ligation of gel-purified <1> to 1-2J or 2-22P, with vector 1-3A, to make <4> or <5> respectively (same <4> and <5> as designed before)

September 5 (Sun)

1. Heat inactivation of yesterday's ligation mixes at 80°C for 20min
2. Transformation of <4>, <5>

２．トランスフォーメーション 　　②のコンピ 50μl＋ライゲーションDNA2μl001+1-2J+1-3A

                                                                  001+2-22P+1-3A  
     Cのプレートに植える

　　 37℃ incubate 11:40~

information 001の代わりになるpromoter+RBSですが、一応パーツ内にありました。 2-10F 2-10H どちらもLac1ではないですが constitutive ON　なのでよければtransformationして下さい。