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From 2010.igem.org

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

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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 (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

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

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

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	(" ")

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

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°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

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

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

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 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

ID	Part Name	Resistance	Description
1-1K	<bbpart>BBa_J63010</bbpart>	A	Protein fusion vector (Silver standard)
1-1I	<bbpart>BBa_J63009</bbpart>	A	Low copy protein fusion vector (Silver standard)
3-3G	<bbpart>BBa_K157013</bbpart>	A	15aa glycine-serine linker (Freiburg standard)

Table 7

MiliQ water	1 liter
Bacto tryptone	20.0g	2%
Bacto yeast extract	10.0g	1%
Glucose	20.0g	2%

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

ID	Part Name	Resistance	Description
1-12D	<bbpart>BBa_E2030</bbpart>	K	yeast-optimized EYFP
1-12B	<bbpart>BBa_E2020</bbpart>	K	yeast-optimized ECFP
3-2K	<bbpart>BBa_K165001</bbpart>	A	yeast GAL1 promoter
1-7D	<bbpart>BBa_J63006</bbpart>	A	yeast 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

ID	Part Name	Resistance	Description
2-10F	<bbpart>BBa_K081005</bbpart>	A	constitutive promoter from combinatorial library + RBS
2-10H	<bbpart>BBa_K081006</bbpart>	A	lambda 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

ID	Part Name	Resistance	Description
A01	pTPG01-1	A	plasmid pTrc99A with pgs genes inserted
A02	pTPG01-2	A	''
A03	pBSGR3	K	glutamine 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 (012, 013) 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. Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis
   • (RESULTS?)
2. PCR test
   • re-cloning of beta-glucosidase from F1 (received from Edinburgh team) into Silver standard-compatible format
   • cloning of pgsC from A01
   • note: primers were misdesigned but PCR performed anyway to confirm whether sequences were complementary, and to identify PCR parameters
3. Gel electrophoresis of PCR products
   • (RESULTS?)
4. Gel purification of PCR product from F1
5. Restriction digest of PCR product (F1) and 1-5A, both with EcoRI, SpeI
6. Gel electrophoresis of PCR product from A01
   • band visible around 400~500bp, which was correct length -> PCR conditions identified!
7. Transformation of A01, A02, A03
8. Transfer of 012, 013, 014 to solution culture
   • RFP expressed from vector plasmids on 012, 013 plates during refrigeration period, so selection of colonies with inserts now possible

September 14 (Tue)

1. Miniprep of A02, 012, 013, 014
2. Restriction digests
   • 012, 013, 014 with EcoRI, PstI
   • A02 with EcoRI
3. Gel electrophoresis of digests
   • A02 was discarded (bad size?)
   • 012~014: digestions repeated with 1μl each of EcoRI, PstI (previous digestions were 0.5μl each)
   • gel electrophoresis of repeat digestions again showed bad sizes for all parts
     • 1-13D terminator part is bad? -> cut check of 1-13D
     • failure to inactivate restriction enzymes before ligations? -> re-ligation
4. 1-13D cut check with XbaI, PstI
5. Re-ligation (3A assembly)
   • previously digested 009, 008, 011 inactivated at 80°C for 20min before ligation
   • 012: 009 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
   • 013: 008 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
   • 014: 011 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/12)
6. Transformation of ligated products (012~014)
7. Gel electrophoresis of yesterday's restriction digests of 1-5A and PCR product from F1 followed by their ligation (1-5A as vector, PCR product as insert)
8. Transfer of A01, A02, A03 to LB liquid culture medium supplanted with 25μl of 2M glucose to prevent leaky expression from Lac promoter (may be affecting growth)
9. Received additional cellulase parts from Karita-sensei: Cel5, Xyn10, Cel44, Man26, Cel8
   • plasmid DNA resuspended in 10μl MiliQ water each
   • transformation with 2μl DNA solution

September 15 (Wed)

1. Miniprep A02, A03 (E. coli failed to grow in A01)
2. Cut check of A02, A03 with EcoRI
   • A02 is ok
3. Transformation of 3-22G
   • (INFO?)
4. PCR cloning of pgsC from A02
5. Colony check & transfer of cellulase parts from Karita-sensei to solution culture
6. Preparation of lysis buffer for yeast genome DNA extraction according to [insert reference]

Component	Volume Added	Final Concentration	Triton X-100	100μl	2%
10%SDS	500μl	1%
5M NaCl	100μl	100mM
20mM Tris-HCl(ph8.0)	2.5ml	10mM
0.5M EDTA(ph8.0)	10μl	1mM
dH2O	1790μl
TOTAL	5ml

1. PCR to clone pgsA, pgsB by 'Megaprimer' method using NEB Phusion polymerase

September 16 (Thu)

1. Gel electrophoresis to verify yesterday's PCR results
   • pgsA megaprimer: 750bp -> OK
   • pgsB megaprimer: 170bp -> OK
   • pgsC: 450bp -> very faint band?
2. Gel extraction of pgsA, pgsB megaprimers
3. 2nd step of megaprimer PCR for pgsA, pgsB
   • failure; possible causes:
     • short annealing step?
     • low denaturation temperature?
     • mistakes in procedure?
4. PCR
   • pgsC using correct (redesigned) primers
   • pgsA, B megaprimer 2nd step repeat using reaction mix composition modified from OpenWetWare
   • gel purification of PCR products
5. Miniprep of BglX (temporary designation for part cloned from F1 with faulty primer), Cel5, Cel8, Cel44, Man26, Xyn10
6. Cut check of miniprepped parts with XbaI, PstI
7. Yeast genome DNA extraction (detailed protocol will be provided elsewhere) according to [reference article]
8. Gel electrophoresis
   • yeast genome DNA
   • PCR products
     • pgsC -> OK
9. Restriction digest
   • pgsC (PCR product) with EcoRI, PstI
   • 1-1C, 1-3A, 1-5A (vectors) with EcoRI, SpeI

September 17 (Fri)

1. PCR cloning of ADH1 terminator from yeast genome DNA
   • 4 simultaneous attempts with varying template concentration, thermocycle settings & timing of polymerase addition (before or after initial denaturation)
   • gel electrophoresis showed no PCR product obtained for any of the reactions -> annealing temperatures were too stringent?
2. PCR of ADH1 terminator (repeat)
   • annealing temperature was lowered from 70°C to 60°C
   • PCR failed again -> possible RNA contamination?
3. PCR of ADH1 terminator (2nd repeat)
   • genomic DNA treated with RNase (0.1μl added to 1μl genome DNA; incubation at 37°C for 15min) before using as template
4. Gel electrophoresis of yesterday's digests: pgsC, 1-1C, 1-3A, 1-5A
5. Miniprep of 013 and 3-22G
   • 012, 014 culture solutions have turned red -> picked-up colonies had RFP-carrying vectors, not ligated plasmids; discarded!
6. Cut check of 3-22G with XbaI, PstI; 013 with EcoRI, PstI
   • (RESULTS?)
7. Ligations
   • 018: 3A assembly with 004 as upstream, Fcex as downstream, 1-1C as vector
   • 019: pgsC as insert, 1-1C as vector (cut at?)
8. PCR cloning of XynA CBM, pgsA
9. Colony pick-up & transfer to solution culture: 006, 007 (9/10 ligation/transformation) more plasmids needed?
10. Transformation of 004, 005, 008, 009, 018, 019

September 18 (Sat)

1. PCR cloning of pgsA by overlap extension megaprimer method doesn't seem to work so well
2. Gel electrophoresis of PCR product (after overlap step) of pgsA
   • correct band seems to be obtained
3. Gel extraction followed by restriction digest of PCR product with EcoRI, PstI
4. Miniprep of 006, 007
5. Restriction digest with EcoRI, PstI followed by gel electrophoresis
6. PCR of pgsB (1st fragment for overlap extension)
   • tried 2 times but couldn't get amplification product!
7. Transfer to solution culture: 004, 005, 008, 009, 018, 019

September 19 (Sun)

1. PCR of pgsB (repeat)
   • again, no product :(
2. PCR of pgsB (4th attempt, including yesterday's)
   • no product
3. Miniprep of 004, 005, 008, 009, 018, 019
4. Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI
5. Gel electrophoresis of pgsA, 004, 005, 008, 009, 018, 019
   • apart from 018 & 019, all are parts digested before; used here to compare with new parts 018, 019
   • (RESULTS?)
6. PCR of pgsB 1st fragment (5th attempt)
   • (RESULTS?)
7. PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension
8. PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension
9. PCR: Phusion activity check using BglX as template & the primers that generated it
10. PCR: pgsB template check using the outermost primers

September 20 (Mon)

1. PCR

1. PCR tube5 control 5xphusion HF Buffer 4μl 10mM dNTP 0.4μl primer 1μl control template DNA 0.8μl phusion 0.2μl H2O 13.6μl total 20μl thermal cycle 98℃ 1min 98℃ 5sec 60℃ 15sec 72℃ 2min30sec 25cycle (boldface) 72℃ 10min 4℃　　　∞

tube1

GC Buffer 4μl dNTP 0.4μl 10xprimer Mix 2μl template (10xA02) 1μl phusion 0.2μl H2O 12.4μl total 20μl

tube2

HF Buffer 4μl dNTP 0.4μl 10xprimer Mix 2μl template (10xA02) 1μl phusion 0.2μl MgCl2(1mM) 4μl H2O 8.4μl total 20μl

thermal cycle 98℃ 1min 98℃ 5sec 40℃ 30sec 72℃ 50sec 30cycle (boldface) 72℃ 10min 4℃　　　∞

control がよくとれている→phusionに間違いなし template 、primerに問題あり？ thermal cycle・反応条件に問題あり？

2. PCR for checking primer　primer のcheckのためのPCR

PCR
         HF Buffer                         32μｌ
         10mM dNTP                     3.2μｌ
          Template(×10)                8μｌ
             Phusion                         1.6μｌ
             DSW                            112μｌ
            total                               156.8μｌ

(ⅰ)

rev4

fwd4

(ⅱ)

rev3

fwd3

(ⅲ)

rev2

fwd2

(ⅳ)

pgsB

rev1

Saka

1

3

5

7

Yasumoto

2

4

6

8

PCR（ⅰ）　 BlockA

         5xPhusion HF Buffer         4μｌ
          10mM dNTP                    0.4μｌ
          Template                          1μｌ
            primer    rev4                 0.2μｌ
            primer    fwd4                0.2μｌ
             Phusion                         0.2μｌ
             DSW                              14μｌ
            total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 65℃ 30sec 72℃ 30sec 35cycle(boldface) 72℃ 10min 4℃　　　∞

PCR（ⅱ）　

         5xPhusion HF Buffer         4μｌ
          10mM dNTP                    0.4μｌ
          Template (x10)                  1μｌ
            primer    rev3                 0.2μｌ
            primer    fwd3                0.2μｌ
             Phusion                         0.2μｌ
             DSW                              14μｌ
            total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 70℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞

PCR（ⅲ）　

         5xPhusion HF Buffer         4μｌ
          10mM dNTP                    0.4μｌ
          Template (x10)                  1μｌ
            primer    rev2                 0.2μｌ
            primer    fwd2                0.2μｌ
             Phusion                         0.2μｌ
             DSW                              14μｌ
            total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 70℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞

PCR（ⅳ）　 BlockB

         5xPhusion HF Buffer         4μｌ
          10mM dNTP                    0.4μｌ
          Template                           1μｌ
            primer    pgsB                 0.2μｌ
            primer    rev1                0.2μｌ
             Phusion                         0.2μｌ
             DSW                              14μｌ
            total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 60℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞

Check PCR fragments by each primers. 各primerによる断片作成チェック ～primer erratum primerの正誤確認～ 98℃ 30sec 98℃ 10sec X℃ 30sec 72℃ 30sec 35cycle (boldface) 72℃ 10min 4℃　　　∞ ～Mix composition～

         5xPhusion HF Buffer         4μｌ
        10mM dNTPs                     0.4μｌ
          Template(×10)                 1μｌ
            primer    1                      0.2μｌ
            primer    2                      0.2μｌ
            primer    3                      0.2μｌ
            primer    4                      0.2μｌ
            polymerase                    0.2μｌ
             DSW                            13.6μｌ
            total                                 20μｌ

　 8. Electrophoresis Apply all sample 全量アプライ(dye 2μｌ、sample　20μｌ、Ladder　10μｌ) 1%Agalose gel、100V　20min

8   7   6   5   4   3   2   1   Ladder

8 , 7→1       6 , 5→2        2 , 1→3

Elution of each sample from gel and purification

3. pgsB 2nd PCR +再々々々 1st PCR

          HF Buffer                          4μｌ
          dNTP                               0.4μｌ
            primer    pgsB              0.2μｌ
                          rev2                0.2μｌ
            tube1                             0.2μｌ
              tube2                             0.2μｌ
             Phusion                         0.2μｌ
             DSW                              14.6μｌ
            total                                 20μｌ

protocol1
98℃   1min
94℃   15sec
50℃   30sec   
74℃   30sec        下３つを2cycle
       　↓flanking  primer3
94℃   15sec
68℃   30sec
72℃   1min      上３つを25cycle
72℃   10min
4℃　　　∞

protocol2   tube8  tube7
98℃   1min
98℃   10sec
50℃   30sec   
70℃   30sec        下３つを2cycle
       　↓Hauking  primers
98℃   10sec
70℃   30sec
68℃   1min      上３つを25cycle
72℃   10min
4℃　　　∞

　　　　　↓ 　　Electropjoresis

4. Electrophoresis dye 2μｌ、Sample　5μｌ、Ladder　4μｌ 1%Agalose gel、100V　20min

PCR1 PCR2 PCR3 Ladder

5. pgsB 1st PCR (part)

    Mix composition
         5xPhusion HF Buffer         4μｌ
        10mM dNTPs                     0.4μｌ
          Template                          1μｌ
            primer    rev                   0.2μｌ
            primer    fwd                   0.2μｌ
            phusion                         0.2μｌ
             DSW                            14μｌ
            total                                 20μｌ

thermal cycle 98℃ 30sec 98℃ 10sec 74℃ 30sec 72℃ 10sec 35cycle(boldface) 72℃ 10min 4℃　　　∞

6. To liquid culture

004(C), 005(C)  ,006(K)  ,007(K)  008(K)  ,009(K)  ,019(A)

To LB medium 37 ℃ incubate 18:20~

New parts 020(A)

              pgsA

7. Ligation

   pgsA  (Eco,Pst)d                    2μl
  1-1C  (Eco,Pst)d                     2μl
  10×T4 DNA ligase Buffer         2μｌ
  T4 DNA ligase                         1μｌ
   H2O                                       13μｌ
  total                                        20μｌ
       room  temp  10min  Ligation
       80℃　 deactivtion by heat   20min

8. Transformation Competent cell②50μl＋LigationLigation product 2μl Inoculte A plate and incubt at 37oC 2：00～

9. Electrophoresis All PCR product＋dye 2μｌ、Ladder　10μｌ 1%Agalose gel、100V　25min

tube8 tube7 tube6 tube5 Ladder tube2 tube1 Ladder

tube1, 2 は300bpあたり tube5, 6, 7, 8 は740bpあたりのバンドを切り出し gel purification

10. Electrophoresis to check gel purification dye 2μｌ、sample 10μｌ、Ladder　10μｌ 1%Agalose gel、100V　20min

tube8 tube7 tube6 tube5 tube2 tube1 Ladder