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	===August 25 (Wed)===		===August 25 (Wed)===
	# Miniprep of parts in solution culture: 2-2O, 3-11I, F1		# Miniprep of parts in solution culture: 2-2O, 3-11I, F1
-	Cut check of 3-11I & F1 with EcoRI, SpeI	+	# Cut check of 3-11I & F1 with EcoRI, SpeI
-		+	#* (RESULTS?)
-	3.電気泳動	+
-	dye 2μl,サンプル 10μl,Ladder 2μl    1% Agalose gel,100V 30min,EtBr 40min	+
-	Ladder,3-11I d,F1 d	+
-		+

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Revision as of 11:55, 5 October 2010

Contents 1 Notebook 1.1 July 29 (Thu) 1.2 July 31 (Sat) 1.3 August 2 (Mon) 1.4 August 3 (Tue) 1.5 August 4 (Wed) 1.6 August 5 (Thu) 1.7 August 6 (Fri) 1.8 August 7 (Sat) 1.9 August 8 (Sun) 1.10 August 9 (Mon) 1.11 August 10 (Tue) 1.12 August 11 (Wed) 1.13 August 16 (Mon) 1.14 August 17 (Tue) 1.15 August 18 (Wed) 1.16 August 19 (Thu) 1.17 August 20 (Fri) 1.18 August 21 (Sat) 1.19 August 22 (Sun) 1.20 August 23 (Mon) 1.21 August 24 (Tue) 1.22 August 25 (Wed)

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:

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

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

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	(" ")

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

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)

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:

ID	Part Name	Resistance	Description
2-4A	<bbpart>BBa_J63005</bbpart>	A	yeast ADH1 promoter
F1	N/A	A	beta-galactosidase from Edinburgh team
F2	N/A	C	RBS + F1
F3	N/A	C	Lac promoter + RBS + F1

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

ID	Part Name	Resistance	Description
2-2O	<bbpart>J63003</bbpart>	A	yeast 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?)