"

From 2014.igem.org

Lab member

Yoshikawa,Nakashima

Contents

Making Plate Culture

Ampicillin *10
Chloramphenicol*6

Lab member

Yoshikawa,Nakashima

Contents

Making DW 100mL

TF

4-17F(A-1), 4-4E(A-2), 3-19O(A-3), 3-4G(A-4), 4-1N(A-6), 3-3F(A-8), 4-13L(B-10)

Lab member

Yoshikawa,Nakashima

</p>

Contents

A-2:No colony

TF

A-2(recovery)

preculture

A-1, A-3, A-4, A-6, A-8, B-10,Escherichia coli JM109(for competent cell)

Lab member

Yoshikawa,Nakashima

Contents

A-2:There are something like colonies.

Making glycerol stock/miniprep

A-1, A-3, A-4, A-6, A-8
All samples:consentration is low.
B-10:disposed

making reagent

LB: 500mL
50mM CaCl2: 400mL
50mM CaCl2/ 20% glycerol: 200mL

preculture

A-1 #1
A-2 #1 #2
A-3 #1
A-4 #1
A-6 #1
A-8 #1
B-10 #1 #2
E. coli JM109（for competent cell）

1. colony number

Lab member

Yoshikawa,Nakashima

Contents

miniprep

A-1,A-3,A-6:from culture 2.5ml OK
A-4,A-8:from culture 1.5ml OK
A-2#1:made glycerol stock. Low consentration.
B-10#1:Low consentration.
A-2#2,B-10#2:did not grow

Making competent cell

100uL*120

making reagent

（2000×）Ampicillin 3ｍL　（100mg/mL）

Lab member

Yoshikawa,Nakashima

Contents

Cut check

A-4 (81ng/μL):ES Cut,XP cut.Checked in 1.5h, 2h, 2.5h, 3h:OK
Left:1kbp Ladder/A-4(Control)/ES 1.5h/ES 2h/ES 2.5h/ES 3h
Right:1kbp Ladder/A-4(Control)/XP 1.5h/XP 2h/XP 2.5h/XP 3h
<img src=""/>

TF

We measured cfu of competent cell we made.
We used Efficiency Kit （RFP Construct on pSB1C3）in distribution Kit.

TF

50pg, 20pg, 10pg, 5pg

competent cell:25uL

Sprinkling:100uL

preculture

B-10 #3, A-4

</p>

Lab member

Yoshikawa,Nakashima

Contents

plate check:Did not grow.

miniprep

A-4, B-10 #3(made glycerol stock):OK

Making plate

200ml,CP*10

Lab member

Nakashima

Contents

digestion,gel extraction

</p>

A-6 SP, B-10 XP：disposed
100bp Ladder, A-6 SP, B-10 XP, NC
<img src=""/>
(wrote at 0731
A-6
band near 2kbp:SP cut or single cut
band near 1.5kbp:Plasmid)

</p>

colony PCR

A-6 #1, B-10 #1
1kbp Ladder, A-6, B-10, NC
<img src=""/>
A-6:OK,B-10:wrong

Measuring cfu

competent cell:50uL

Lab member

Yoshikawa,Nakashima

Contents

Plate check:All Plates did not grow.

TF

B-10 by electroporation.

Measuring cfu

A-1:13,65,130pg

Electrophoresis check

1kbp Ladder, A-6(Plasmid), NC
<img src=""/>
There is band in 1500kbp,so 1500kbp in 0728 may be rest of cutting.

No contamination in Dye.

</p>

Lab member

Yoshikawa,Nakashima

Contents

colony check

cfu:Did not grow
B-10:One colony

Cut check

We checked whether A-6 band in 0728 was rest of cutting.

A-1 SP cut:checked on different times.

colony PCR

B-10(from Plate made in 0729)
1kbp Ladder, 1.5h, 2h, 2.5h, B-10(colony PCR), NC
<img src=""/>
band near 800bp:RFP between Spe1 site and Pst1 site of A-1.
band near 2kbp:Backbone cut by SP cut
band near 3kbp:rest of cutting

1.5h:incompletely cut
2h,2.5h:OK
We decided 2h for digestion.
B-10:OK

preculture

B-10

Lab member

Yoshikawa,Nakashima

Contents

miniprep

B-10 #1（from 140729 Plate）(made glycerol stock)

digestion,gel extraction

A-6 SP, B-10 XP

1kbp Ladder, A-6, B-10
<img src=""/>
B-10:incomplete cut
others:OK

ligation

C-7 (A-6 SP + B-10 XP)

Lab member

Yoshikawa,Nakashima

Contents

TF

C-7（Chemical & EP）

ligation Check

PCR reaction primed with Universal Primer, using ligation products as template.
<img src=""/>
OK!

Lab member

Yoshikawa,Nakashima

Contents

Plate check

No colony

digestion

A-6 SP, B-10 XP

Electrophoresis

1kbp Ladder, A-6, B-10
<img src=""/>
Incomplete cut.Disposed.

preculture

B-10

Lab member

Yoshikawa,Nakashima

Contents

miniprep

B-10　→Sample Lost!　Bye bye, GFP!

Cut check

SP(A-1), XP(A-1), EX(A-4), ES(A-4)
2h, 2.5h, 3h

Elrctrophoresis

1kbp Ladder, A-1, A-1 SP(2h, 2.5h, 3h), A-1 XP(2h,2.5h, 3h), A-4,

A-4 EX(2h, 2.5h 3h), A-4 ES(2h, 2.5h, 3h), None, 1kbp Ladder

<img src=""/>

Incomplete cut.

preculture

A-1, A-4, B-10

Lab member

Yoshikawa

Contents

miniprep

A-1, A-4, B-10

digestion, gel extraction

A-6 SP, B-10 XP

1kbp Ladder, A-6, B-10
<img src=""/>
A-6:OK
B-10:Incomplete cut
<img src=""/>

We chenged restriction enzyme.
B-10 XP
<img src=""/>
OK!
<img src=""/>

making gel

200ml

ligation

C-7（A-6 SP + B-10 XP）

TF

4-11L, C-7

Lab member

Yoshikawa,Nakashima,Tara,Itoh,Tsukada

Contents

colony PCR

C-7
<img src=""/>
<img src=""/>
All bands are self ligation of backbone.

digestion, gel extraction

A-6 SP, B-10 XP

1kbp Ladder, B-10, A-6
<img src=""/>

ligation

C-7 (A-6 SP + B-10 XP)

Cut check

(O/N)

（enzyme-buffer) S-B, S-H*, S-M*, P*-FD, E-FD

• Takara's product
  

</p>

Lab member

Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa

Contents

Cut check sample Electrophoresis

1kbp Ladder, S-B, S-H*, S-M*, NC, E-FD, P*-FD
<img src=""/>
FD buffer is appropriate for SP cut.

digestion, gel extraction

A-6 SP, B-10 XP
<img src=""/>
<img src=""/>
OK!

ligation

C-7 (A-6 SP + B-10 XP)

TF

C-7,4-11L(culture in test tube)

overlap extension PCR →PCR clean up

1+2
anealing 57℃,extension 10 sec, 30 cycle.

100bp Ladder, Pecf11, Pecf20, crRBS, taRNA
<img src=""/>
OK!(low concentration)

making gel

200ml

</p>

Lab member

Yoshikawa

Contents

Plate and test tube check

4-11L:OK!mixed with glycerol, and conserved in -80℃.
C-7:OK! conserved in refrigerator.

Lab member

Yoshikawa,Nakashima,Yamanaka,Nakamura

Contents

colony PCR

C-7 #1~11

1. 1~4, NC
   

<img src=""/>

1. 5~11,NC
   

<img src=""/>

1. 10 is OK!
   

overlap extension PCR →PCR clean up

Pecf11, Pecf20, crRBS, taRNA (1+2)+3,PCR product
1kbp Ladder, Pecf11(1+2+3), Pecf20(1+2+3), crRBS(1+2+3)

taRNA(1+2+3), Pecf11(all), Pecf20(all), crRBS(all), taRNA(all)

<img src=""/>

Making Plate

Ampicillin*20

preculture

A-6, B-10, C-7 #10

Lab member

Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa

Contents

miniprep

A-6, B-10, C-6 #10 (made glycerol stock)

digestion →gel extraction

A-8 EX, C-6 ES, pSB1A2(A-1) XP *2 sample, A-9 linear XP
A-10 linear XP, A-7 linear XP, B-1 linear XP

1kbp Ladder, A-1 XP, A-1 XP, A-8 EX, C-6 ES
<img src=""/>
C-6:wrong
A-1:OK!

colony PCR

4-11L
<img src=""/>
OK!

ligation →TF

A-9, A-10, A-7, B-1

Making　reagent

LB 800ml

Lab member

Yoshikawa,Nakashima,Tara,Nakamura,Takemura,Tsukada

Contents

miniprep

4-11L（made glycerol stock）

digestion→ gel extrction

A-8 EX, C-6 ES, A-6 SP, 4-11L XP

1kbp Ladder, A-6 SP, A-8 EX, C-6 ES, 4-11L XP
<img src=""/>
<img src=""/>
C-6:Incomplete cut
Backbone of 4-11L said to be pSB1A2, but ,actually,may be pSB1AK3.

ligation→TF

D-7 (4-11L XP + A-6 SP), D-7(C-6 ES + A-8 EX)

colony PCR

A-7 #1~2, A-9 #1~4, A-10 #1~4, B-1 #1~2 ,100bp Ladder
<img src=""/>
A-7 #1, A-10 #4：OK!

preculture

A-8, C-6, A-7 #1, A-10 #4

Lab member

Yoshikawa,Nakashima,Tara,Takemura,Nakamura

Contents

miniprep

A-7 #1（made glycerol stock）, A-10 #4（made glycerol stock）, A-8, C-6

overlap extension PCR

Pecf 11, Pecf 20, crRBS, taRNA
(35 cycles, annealing in 59℃)

digestion → gel extranction

A-7 SP, 4-11L XP, pSB1A2 (B-10), A-7 linear XP
A-9 linear XP, A-10 linear XP, B-1 linear XP

1kbp Ladder, A-7 SP, 4-11L XP
<img src=""/>
<img src=""/>
OK!
A-10 XP, 100bp Ladder
<img src=""/>
OK!(low concentration)

100bp Ladder, pSB1A2(B-10), pSB1A2(B-10), A-7, A-9, B-1
<img src=""/>
<img src=""/>
Incomplete cut of pSB1A2 is weigh on our mind.

gel making

2% 25mL
1% 200mL

colony PCR

D-7(Ampicillin), D-7(Chloramphenicol)

1kbp Ladder, D-7(Amp) #1~4, D-7(CP) #1~4
<img src=""/>

ligation → TF

D-6 (A-7 SP + 4-11L XP), A-7 (linear XP + pSB1A2 XP)
A-9, A-10, B-1 (linear XP + pSB1A2 XP)

Lab member

Yoshikawa,Nakashima,Tara,Takemura,Nakamura

Contents

miniprep

D-7 #3 (made glycerol stock)

digestion → gel extraction

A-10 SP, D-7 XP
<img src=""/>
<img src=""/>

colony PCR

A-7 #1~4, A-9 #1~4, A-10 #1~4, B-1 #1~4, D-6 #1~4
<img src=""/>
A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4：OK!

D-6
<img src=""/>
OK!

ligation → TF

E-18 (A-10 SP + D-7 XP)

preculture

A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1

Lab member

Yoshikawa

Contents

miniprep

A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1
(made glycerol stock)

Lab member

Yoshikawa,Hiura

Contents

digestion → gel extraction

A-1 SP, A-2 SP, A-3 SP, D-6 XP *2, A-7 #3,4, B-10
<img src=""/>

sequence

A-7, A-7 #3,4, A-10, A-10 #1,2,4, B-1 #1,2,4
C-6 F, C-6 R, D-7 F, D-7 R, D-6 F, D-6 R

colony PCR

E-18 #1~3
<img src=""/>
OK!

ligation → TF

E-14 (A-3 SP + D-6 XP)
E-15 (A-1 SP + D-6 XP)
E-16 (A-2 SP + D-6 XP)

preculture

E-18 #1

Lab member

Yoshikawa,Nakamura,Yamanaka,Yoshikawa(Fresh)

Contents

miniprep

E-18 (made glycerol stock)

digestion

A-8 XP, B-1 #1,2,4 SP
→Today, sequences of these dna proved to be wrong, so we disposed them.

</p>

making gel

2% gel:25ml

colony PCR

C-5, E-14, E-15,E-16

sequence

A-7:RBS
A-7 #3:none
A-7 #4:none
A-10:OK
B-1: all none

Lab member

Yoshikawa,Nakamura,Tsukada,Yamanaka,Itoh

Contents

PCR

sigma11, sigma20, anti11, anti20

TF

1-21P

Lab member

<p>Yoshikawa,Nakashima

</p>

Contents

digestion → gel extraction

pSB1C3(A-4), A-6, B-3(We did not extract.), A-6'(NEB buffer)
<img src=""/>

colony PCR

A-2, A-3, A-4, 1-21P, NC
<img src=""/>

ligation → TF

B-3, C-2, C-2'

preculture

1-21P

Lab member

Yoshikawa,Nakamura,Nakashima

Contents

miniprep

1-21P(made glycerol stock)

colony PCR

B-3, C-2, C-2
<img src=""/>

digestion → gel extraction

1-21P SP, D-7 XP, C-6 E, C-6 P, C-6(NC)
<img src=""/>
<img src=""/>

ligation → TF

K-1(1-21P SP + D-7 XP)

preculture

B-3 #1,4, D-7, C-2 #1,3, C-2' #3,4

Lab member

Yoshikawa

Contents

miniprep

D-7, B-3 #1,4, C-2 #1,3, C-2' #3,4

Lab member

Nakashima,Nakamura,Yamanaka,Hiura

Contents

digestion → gel extraction

A-8 EX *2, B-3 #1 ES, B-3 #4 ES, C-2 #1 ES, C-2 #3 ES
<img src=""/>
<img src=""/>

Plate　making

Ampicillin*10, Chloramphenicol*20

colony PCR

K-1
<img src=""/>

1. 1,4:OK!
   

sequence

B-3 #1,4, C-2 #1,3, C-2' #3,4, E-18, A-2, A-3

ligation → TF

D-3 1 (A-8 EX + C-2 #1 ES), D-3 2(A-8 EX + C-2 #3 ES)
C-10 1(A-8 EX + B-3 #1 ES), C-10 2(A-8 EX + B-3 #4 ES)

preculture

A-2, A-8, K-1 #1, C-2 #1,3, B-3 #1

Lab member

Yoshikawa,Nakashima,Yamanaka,Takemura,Tsukada

Contents

miniprep

K-1(made glycerol stock), A-2, A-8, B-3 #1, C-2 #1, C-2 #3

colony PCR

D-3 (1), D-3 (2), C-10 (1), C-10 (2)
<img src=""/>
OK!(C-10(1)#4 may be a little shorter.)

digestion → gel extraction

K-2 linear EP, K-3 linear EP, K-4 limear EP, K-5 linear EP, pSB1C3 (A-4) EP
<img src=""/>
We forgot to take the photo before we sliced the band.

ligation → TF

K-2, K-3, K-4, K-5

preculture

D-3 #1, C-10 #1

sequence

A-2:E-X-S-S-Pconst(weak)-S-P wrong

sequence

A-3:OK
B-3 #1:OK
B-3 #4:OK
C-2 #1:OK
C-2 #3:OK
C-2' #3:point mutation *2
C-2' #4:OK
E-18:OK

Lab member

Yoshikawa,Nakashima,Yoshikawa(fresh),Itoh,Tara,Nakamura

Contents

miniprep

D-3, C-10(made glycerol stock)

digestion → gel extraction

A-1 SP, A-3 SP, A-10 SP, D-3 XP *2
<img src=""/>
<img src=""/>
D-3 is a little strange.Contamination?

colony PCR

K-2~5 #1~4

• 100bp Ladder
  

<img src=""/>
500bp band:self ligation of backbone(A-4 200bop)
300bp band:If this band is blank vector, this band(EP cut) must be shorter than 300bp.

So, this band is OK!.

→K-2 #4, K-3 #1, K-4 #1,2, K-5 #2,3,4:OK!

ligation → TF

E-5(A-3 SP + D-3 XP), E-6(A-1 SP + D-3 XP), E-20(A-10 SP + D-3 XP)

preculture

E-15
D-3, C-10(Recovery)
K-2 #4, K-3 #1, K-5 #2

Remarks

<img src=""/>
We observed that pCMV expressed in Escherichia.coli.
Left:pCMV-GFP
Right:pConst(strong)-GFP
We may be able to carry out the characterization of pCMV and meet the gold medal requirement(parts implovement).

Lab member

Yoshikawa,Nakashima,Itoh,Tara,Tsukada,Yamanaka

Contents

miniprep

K-2~5, E-23(made glycerol stock)
C-10, D-3

digestion →gel extraction

K-2 EX, K-3 EX, K-4 EX, K-5 EX, E-23 EP, C-6 ES *2, pSB1C3(A-4) EP, pSB1C3(A-4) XP
<img src=""/>
<img src=""/>
→pSB1C3 XP:keep in freezer

overlap extension PCR

A-7, A-9, B-1, B-2, D-8, D-9

check & colony PCR

check:E-5, E-6
colony PCR:(E-5 #1~4, E-6 #1~4, NC), A-7, A-9, D-8, B-1, B-2, D-9
<img src=""/>
A-7, A-9, B-1, D-8:OK!
B-2, D-9:Needs retry in higher concentration.
E-5 #2~4, E-6:OK!

ligation → TF

E-23'(E-23 EP + pSB1C3 EP), L-1~4(C-6 ES + K-2~5 EX)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Tsukada,Yamanaka

Contents

miniprep

E-5(made glycerol stock)
A-4, C-6
E-6:No colony

digestion →gel extraction

A-7 linear XP, A-9 linear XP, A-10 SP, B-1 linear XP, D-3 XP, D-8 linear XP, E-5 SP, E-18 XP
<img src=""/>
<img src=""/>

PCR

B-2, D-9(retry)

check & colony PCR

E-23' #1~4, B-2, D-9
<img src=""/>
L-1~4
<img src=""/>
E-23 #1, L-1 #2,4, L-2 #4, L-3 #1~3, L-4 #2,4:OK!
PCR:failed

sequence

order

E-23, K-1, K-2~5

</p>

ligation → TF

A-7, A-9, B-1, D-8( linear XP + pSB1C3 XP)
F-2 (E-18 XP + E-5 SP), E-20(A-10 SP + D-3 XP)

preculture

A-10, E-6, E-18, E-23', K-3, L-1~4

Lab member

Yoshikawa

Contents

miniprep

A-10, E-18, K-3
L-1~4, E-23', E-6, E-18(made glycerol stock)

PCR

B-2, D-9

Lab member

Nakashima,Itoh,Tara,Tsukada,Takemura

Contents

PCR clean up

B-2, D-9
<img src=""/>
failed

digestion → gel extraction

L-4 ES, E-6 ES, E-18 EX, L-1~3 ES, K-2~5 EX

<img src=""/>
<img src=""/>

Making plate

CP *30

colony PCR

A-7 #1~4, A-9 #1
<img src=""/>
OK!
D-8 #1~4, E-20 #1~4, F-2 #1~4 (100bp Ladder)
<img src=""/>
D-8 #1~4, E-20 #1~4,F-2#1,4:OK!
B-1 #1~4
<img src=""/>
OK!

Ligation

G-5 (E-6 ES + E-18 EX), M-1~4(K-2~5 EX + L-1~4 ES)

</p>

preculture

A-7 #1~4, A-9 #1, B-1 #1~4, D-8 #1~4, F-2, E-20

Lab member

Yoshikawa,Nakashima,Hiura,Tara,Tsukada,Takemura

Contents

miniprep

A-7 #1~4, A-9, B-1 #1~4, D-8 #1~4, F-2, E-2(made glycerol stock)

digestion → gel extraction

A-4 SP, A-7 SP #1, A-7 SP #2, A-9 SP
<img src=""/>
<img src=""/>
A-8XP
<img src=""/>
<img src=""/>

B-1 #1, B-1 #2, B-3 XP, B-10 XP, D-7 XP, D-8 XP, E-20 XP, F-2 SP
<img src=""/>
<img src=""/>
D-8:???

sequence

A-7 #1~4, B-1 #1~4
D-8 #1~4
A-9, A-4, E-5, E-6, E-20

PCR

B-2, D-9

check & colony PCR

M-1~3 #1~4
<img src=""/>
OK!
M-4 #1~4, B-2, D-9, PC(VF2→B-10←VR )
NC, G-5
<img src=""/>
M-4 #1~4,PC(VF2→B-10←VR ):OK!
(M-4 #2~4 is a little longer?)

ligation → TF

C-4(A-7 SP + B-3 XP), C-5(A-7 SP + B-10 XP), E-17(A-9 SP + D-7 XP)

D-1(B-1 SP + D-8 XP), E-21(A-4 SP + D-8 XP), J-4 (F-2 SP + E-20 XP)

preculture

M-1~4 #1, G-5 #1, E-20

We got a result!!

simpler version of assay 1.
Left:Pσ11→GFP generator
Right:Pconst(strong)→σ11 generator & Pσ11→GFP generator
<img src=""/>

Lab member

Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Takemura

Contents

M-1~4, G-5(made glycerol stock of M-3,4)

sequence

A-4: OK
A-7: all OK
A-9: OK
B-1:all OK
D-8#1: NG (2 skip)

    #2; OK

    #3: NG (1 mut.)

    #4; NG (2 mut. 2 skip)

E-5: OK
E-6: OK
E-20:OK

digestion → gel extraction

A-7 #1, 2 SP, C-9 XP, C-10 XP, K-2~4 EX, M-1~4 ES
<img src=""/>
<img src=""/>

colony PCR

C-4 a, C-5 a, D-1 a, E-17
<img src=""/>
C-4 a#2,3, C-5 a, D-1 a, E-17:OK!
E-21 b, J-4
<img src=""/>
J-4:wrong
E-21:a little shorter

PCR

B-2, D-9, B-7, B-2(Taq), D-9(Taq) Taq:positive control
anealing temperature:51℃～61℃(gradient)

ligation → TF

D-5 (A-7 SP + C-10 XP), D-6 (A-7 SP + C-9 XP), N-1~4 (M-1~4 ES + K-2~5 EX)

N-5 (M-1 ES K-3 EX), N-6 (M-3 ES + K-5 EX)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka,Tsukada

Contents

miniprep

E-20, D-1(made glycerol stock)

digestion → gel extraction

pSB1C3 (A-4) XS, pSB1C3 (A-4) XP, A-3 SP, D-1 XP
<img src=""/>
<img src=""/>

PCR check & clean up

D-9 #1,3,5,7,9
<img src=""/>
There are bands in #5,7,9
B-2 1,3,5,7,9,11, B-7.
<img src=""/>
There are bands in B-2 #5,7, B-7.
B-2 4,6, D-9 10,11,12
<img src=""/>
OK!
We decided to clean up B-2 #5, D-9 #11, B-7.

Making Plate

CP *30

digestion

B-2 linear SP, D-9 linear XP, B-7 linear XP

colony PCR

D-5, D-6, N-1, N-2 #1~4
<img src=""/>
D-5, D-6, N-1, N-2 #1,2:OK!
N-3~6 #1~4
<img src=""/>
N-3,N-4#1,3,4,N-5#1,4,N-6:OK!

ligation → TF

E-1 (A-3 SP + D-1 XP), B-2 (B-2 linear XP + pSB1C3 XP), D-9 (D-9 linear XP + pSB1C3 XP)

B-7 (B-7 linear XP + pSB1C3 XP), Emp. (pSB1C3 XS)

miniprep

M-1, M-2, C-4, C-5, E-17, E-21, J-4 (made glycerol stock)
K-2, K-5, C-9

preculture

F-2, D-5, D-6, N-1~6

Lab member

Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka

Contents

miniprep

F-2
N-1~6, D-5, D-6
→N-1, 2, 4, 5. 6 :failed

digestion → gel extraction

A-1 SP, A-3 SP, D-5 XP, D-6 SP, E-20 XP, K-4 EX, N-3 ES, F-2 SP
J-4 E(check)
<img src=""/>
<img src=""/>

ligation → TF

E-11 (A-3 SP + D-5 XP), E-12 (A-1 SP + D-5 XP), E-14 (A-3 SP + D-6 SP)

E-15 (A-1 SP + D-6 XP), O-3 (K-4 EX + N-3 ES)

PCR

B-2, D-9, B-7

colony PCR

B-2, B-7, D-9, E-1　#1~4
<img src=""/>
B-7 #2,3, D-9 #1, E-1 #3,4:OK!
Z-1 #2~4
<img src=""/>

preculture

B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2, K-3, N-1,2,4~6

Lab member

<p>Yoshikawa,Nakashima

</p>

Contents

miniprep

B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2 (made glycerol stock)
N-1,2,4~6, K-3

PCR

B-2, D-9, B-7

check & colony PCR

B-2, D-9, B-7, B-2#5~8, B-7 #2,3,5,6, D-9 #5,6.7,8
<img src=""/>

Lab member

Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka,Tara

Contents

digestion → gel extaction

B-2 linear XP (did not extracted)
N-1,2,4~6 ES
<img src=""/>
<img src=""/>
A-4 SP, pSB1C3 (A-4) XP, A-6 SP, B-7 #2 XP, B-7 #3 XP, D-9 #1 XP, K-2,3,5 EX
<img src=""/>
B-7 #2, D-9 #1:disposed(incomplete cut)
B-7 #3:disposed(a little longer)(This band proved to be correct.9/9 wrote)
<img src=""/>

colony PCR

E-11,12,14,15 #1~4
<img src=""/>
OK!
O-3 #1,2
<img src=""/>

1. 2:OK!
   

PCR

B-2
<img src=""/>
B-7,D-9
<img src=""/>
The band of the longest DNA is OK!

ligation → TF

O-1 (N-1 ES + K-2 EX), O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), O-5 (N-5 ES + K-3 EX)

O-6 (N-6 ES + K-5 EX), B-2 (B-2 linear XP (9/8

PCR

) + pSB1C3 XP)

B-2' (B-2 linear XP + pSB1C3 XP), D-9, B-7

</p>

preculture

D-5, D-6, N-3, E-21, A-3, K-4
E-11 #1, E-12 #1, E-14 #1, E-15 #1, O-3 #2, B-7 #6, D-9 #5,6

Lab member

Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka

Contents

miniprep

E-11,12,14,15, O-3, B-7 #6, D-9 #5,6 (made glycerol stock)
D-5, N-3, K-4, A-3
D-6:did not grow
D-5:diposed(doubt of cross contamination)

digestion → gel extraction

B-2 linear XP (did not extracted)
pSB1C3 (A-4) XP, A-6 SP, A-8 EX, B-7 #6 XP, D-9 linear XP, B-7 linear , E-1 EX, E-14 ES, E-15 ES, O-3 ES
<img src=""/>
<img src=""/>

colony PCR

B-2 #1~3, B-2' #1~4, B-7 #1~3, D-9 #1,2, O-1 #1~4
<img src=""/>
B-2' #1, B-7 #1,2, O-1:OK!
O-2,4,5,6 #1~4
<img src=""/>
O-2,3,4,6,O-5#2,3,4

sequence

B-7 #2: NG
B-7 #3: OK
D-9 #1: NG
E-1: OK
C-4: OK
C-5: OK
D-5: OK
D-6: OK
E-17: OK
E-21: OK
F-2: ?
J-4: OK
N-1: OK
N-2: M-2
N-3: OK
N-4: M-4
N-5: OK
N-6: OK

ligation → TF

D-9 (D-9 linear XP + pSB1C3 XP) *2, B-2 (B-2 linear XP + pSB1C3 XP)*2

P-3 (O-3 ES + A-8 EX), I-1 (E-14 ES + E-1 EX), I-2 (E-15 ES + E-1 EX)

preculture

F-2, D-5,6, D-9 #5,6, E-12, K-4
O-1,2,4,6 #1, O-5 #2, B-2' #1

Lab member

Nakashima,Hiura,Takemura,Yamanaka,Yoshikawa(Fresh)

Contents

miniprep

O-1,5,6, N-2,4, F-2, B-2' #1 (made glycerol stock)
E-12, K-4, D-5
D-6:did not grow

digestion → gel extraction

A-6 SP, A-7 SP, A-8 EX, B-2' XP, B-7 XP
<img src=""/>
<img src=""/>
K-3 EX, K-5 EX, O-1 ES, N-2 ES, N-4 ES
<img src=""/>
<img src=""/>
O-5 ES, O-6 ES
<img src=""/>
<img src=""/>

colony PCR

I-1 #1~4
<img src=""/>
B-2(140909) #1~8, D-9 #1~8
<img src=""/>
B-2 #2, D-9 all, I-1 #2~4, I-2 #1~4:OK!

ligation → TF

O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), P-1 (O-1 ES + A-8 EX), P-5 (O-5 ES + A-8 EX)

P-6 (O-6 ES + A-8 EX), C-1' (B-2' XP + A-6 SP), C-3' (B-2 ' XP + A-7 SP), C-8 (B-7 XP + A-6 SP)

preculture

D-9 #5,6, D-9(140909) #1~4, B-2 (140909) #2, I-1 #2, I-2 #1, D-6 #1

Lab member

Yoshikawa,Nakashima,Nakamura,Hiura,Itoh,Tsukada

Contents

miniprep

D-9 #1~4, B-2 #2, I-1, I-2, D-6 (made glycerol stock)

digestion → gel extraction

A-1 EP, pSB1C3(A-3) EP *2, A-4 SP, A-6 SP, A-7 SP, A-8 EX, A-10 EX, B-2 #2 XP, E-6 EP, E-12 EP
<img src=""/>
<img src=""/>
E-15 EP, E-18 EP, E-20 EP, D-9 #1 XP, O-3 ES
<img src=""/>
<img src=""/>

sequence

B-2' #1, B-2 #2, F-2, E-11, E-12, E-14, E-15, O-1, N-2, O-3, N-4, O-5, O-6, D-9 #1~4

Plate Making

CP *30

colony PCR

C-1', C-3', C-8, O-2, O-4 #1~3
<img src=""/>
C-1', C-3', C-8, O-2, O-4 #1,2:OK!
P-1, P-5, P-6 #1~3
<img src=""/>
→P-1 #1~3, P-5 #2, P-6 #1~3:OK!

ligation → TF

C-1 (A-6 SP + B-2 #2 XP), C-3 (A-7 SP + B-2 #2 XP)

E-22a (A-4 SP + D-9 #1 XP), E-22b (A-4 SP + D-9 #2 XP), P-3 (O-3 ES + A-8 EX)

A-10 CP, A-1 CP, E-6 CP, E-12 CP, E-20 CP, E-15 CP (replace backbone to pSB1C3)

preculture

C-1' #1, C-3' #1, C-8 #1, O-2 #1, O-4 #1, P-1 #1, P-5 #2, P-6 #1, E-12,14,15,18, A-1, O-1,3,5

Lab member

Yoshikawa,Nakashima,Hiura,Itoh,Tsukada,Tara

Contents

miniprep

C-1', C-3', O-2, O-4, P-1, P-5, P-6 (made glycerol stock)
E-12, E-14, E-15, O-1, O-3, O-5, E-18, A-1

digestion → gel extraction

A-6 SP, A-8 EX *2, B-2 XP, C-1' ES, C-3' ES, C-8 ES, K-6 SP, O-2 ES, O-3 ES, O-4 ES
<img src=""/>
<img src=""/>
C-1,3:did not extracted
P-1 XP, P-5 XP, P-6 XP
<img src=""/>
<img src=""/>

colony PCR

A-1 CP, A-10 CP, C-3, E-6 CP #1~3
<img src=""/>
E-12 CP, E-15 CP, E-20 CP, E-22a, E-22b
<img src=""/>
except E-20#1,2:OK!

sequence

B-2' #1: NG
B-2 #2: OK
D-9 #1: NG

   #2: NG

   #3: NG

   #4: OK

O-1: OK
O-3: OK
O-5: OK
O-6: OK
N-2: OK
N-4: OK
E-11: Ok
E-12: OK
E-14: OK
E-15: OK
F-2: OK

ligation → TF

C-1 (A-6 SP + B-2 XP), P-2~4 (O-2~4 ES + A-8 EX), Q-1,5,6 (P-1,5,6 XP + K-6 SP)

ligation

P-3 (O-3 ES + A-8 EX)

preculture

A-4, A-6, A-8, C-3 #1, A-1 CP #1, E-12 CP #1, E-15 CP #1, E-20 CP #3, E-6 CP #1

9,13,2014

</div>

Lab member

Yoshikawa

Contents

miniprep

A-4, A-6, A-8 C-3, E-6 CP, E-12 CP, E-15 CP, E-20 CP, A-1 CP(made glycerol stock)

Lab member

Yoshikawa,Nakashima,Takemura,Nakamura,Yamanaka,Tara

Contents

digestion → gel extraction

A-8 EX, C-3 ES
<img src=""/>
<img src=""/>

colony PCR

C-1 (σ20F, VR)
<img src=""/>
OK!

ligation → TF

D-4 (C-3 ES + A-8 EX) *2 (new and old competent cell)

preculture

C-1 #1

Lab member

Yoshikawa,Nakashima,Hiura,Itoh,Nakamura,Yamanaka

Contents

miniprep

C-1　(made glycerol stock)

colony PCR

D-4, P-2~4
<img src=""/>
A-10
<img src=""/>

Q-1,5,6:confirmed by fluorescence

digestion → gel extraction

A-4 SP, A-8 EX, C-1 ES, D-9 XP, E-18 EP, pSB1C3 (A-4) EP
<img src=""/>

ligation → TF

D-2 (A-8 EX + C-1 ES), E-18 CP (E-18 EP + pSB1C3 EP) *2, E-22 (A-4 SP + D-9 XP)

preculture

D-4, P-2~4, A-10 CP, Q-1,5,6

assay

<img src=""/>
PC (E-23': Pconst (strong)-GFP-d.term)
Experiment (K-1: pCMV-GFP-d.term)
NC (Z-1: pSB1C3)
absorbance:600nm and 395nm Absorbance of 395nm proved not to be able to measure.
We need fluorospectro-photometer.

Lab member

Yoshikawa,Nakashima,Hiura,Takemura,Yoshikawa(Fresh),Yamanaka

Contents

miniprep

D-4, P-2~4, Q-1,5,6, A-10 CP　(made glycerol stock)

digestion → gel extraction

A-1 CP SP, A-3 SP, D-4 XP, K-6 SP, P-2 XP, P-3 XP, P-4 XP
<img src=""/>
<img src=""/>

colony PCR

D-2, E-18 CP, E-22
<img src=""/>

ligation → TF

E-8 *2 (A-3 SP + D-4 XP), E-9 (A-1 CP SP + D-4 XP), Q-2~4 (K-6 SP + P-2~4 XP)

assay

Photo of plate(n=4)
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>

Lab member

Yoshikawa,Nakashima,Hiura,Tsukada,Nakamura

Contents

miniprep

D-2, E-18 CP, E-22
(made glycerol stock)

digestion → gel extraction

A-1 CP SP, A-3 SP, A-9 SP, D-2 XP, E-22 SP, G-5 XP
<img src=""/>
<img src=""/>

colony PCR

E-8, E-9　#1~4
<img src=""/>
Q-2~4 :confirmed by fluorescent

ligation → TF

E-2 (A-3 SP + D-2 XP)
E-3 (A-1CP SP + D-2 XP)
E-19 (A-9 SP + D-2 XP)
H-5 (E-22 SP + G-5 XP)

preculture

E-8,9, Q-2~4

Lab member

Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Nakamura

Contents

miniprep

E-8,9, Q-2~4

digestion → gel extraction

A-9 SP, D-2 XP, E-22 XP, F-2 SP, G-5 SP
<img src=""/>
<img src=""/>

colony PCR

E-2, E-19 #1~4
E-3, H-5 :No colony
<img src=""/>20140919_3IMGP0)

ligation → TF

H-5 (G-5 SP + E-22 XP)
H-4 (F-2 SP + E-22 XP)

preculture

E-2 E#1, E-19 #1

Lab member

Yoshikawa

Contents

miniprep

E-2, E-19

Lab member

Yoshikawa,Nakashima,Takemura,Nakamura,Tara,Yamanaka

Contents

digestion → gel extraction

A-1 SP, D-2 XP, E-2 ES, E-17 EX, E-22 XP, G-5 SP, J-4 SP
<img src=""/>
<img src=""/>

colony PCR

H-4
<img src=""/>
H-4#1,2,3:OK!

ligation - TF

H-5 (G-5 SP + E-22 XP)
J-6 (J-4 SP + E-22 XP)
E-3 (A-1 SP + D-2 XP)
F-1 (E-2 ES + E-17 EX)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka

Contents

digestion, gel extraction

A-1CP SP, D-2 XP, E-22 XP, G-5 SP
J-4 SP(stopped)
<img src=""/>
<img src=""/>

miniprep

H-4(made glycerol stock)

colony PCR

J-6
<img src=""/>
OK!

ligation,TF

H-5(G-5 SP+E22 XP)
E-3(A-1 SP+D-2 XP)

Lab member

Nakashima,Yamanaka,Takemura

Contents

miniprep

F-1,J-6(made glycerol stock)
E-22,D-6
G-6 did not grow.
We disposed J-6.(dropped on the floor)

digestion,gel extraction

A-1 SP,E-2 ES,E-5 ES,E-17 EX,E-18CP EX,E-21 XP,G-5 SP,D-2 XP,F-1 SP,E-19 XP,E-22 XP
<img src=""/>
<img src=""/>

ligation,TF

J-2(F-1 SP+E-1 XP)
H-1(F-1 SP+E-21 XP)
F-3(E-5 ES+E-17 EX)
F-4(E-2 ES+E-18 EX)
E-3(A-1 SP+D-2 XP)
H-5(G-5 SP+E-22 XP)

Lab member

Nakashima,Nakamura,Tara,Tsukada,Yamanaka

Contents

miniprep

A-1CP, J-6

digestion,gel extraction

A-1 SP,D-2 XP,E-5 ES,E-17 EX,E-19 XP,E-21 XP,E-22 XP,F-1 SP,G-5 SP
<img src=""/>
<img src=""/>

ligation,TF

J-2(F-1 SP+E-19 XP)
H-1(F-1 SP+E-21 XP)
F-3(E-5 ES+E-17 EX)
E-3(A-1 SP+D-2 XP)
H-5(G-5 SP+E-22 XP)

Making competent cell

preculture

G-5,E-5,E-17,E. coli JM109

competent cell(made at 0911) check

Ampicillin,Chloramphenicol,non-antibiotics
culture in LB 3ml
LB midium 3ml, as N.C.
culture for 2h.
no TF

result of check

Ampicilln,non-antibiotics:become clouded
Chroramphenicol,LB:did not grow
So,we confirmed ampicillin resistant plasmid exists in competent cell and we disposed it.

Lab member

Yoshikawa,Nakashima,Tara,Yoshikawa(Fresh),Tsukada

Contents

miniprep

G-5,E-5,E-17

digestion

A-1CP SP,D-2 XP,G-5 EP(strange band appeared,disposed),pSB1C3(A-4) EP,A-1 SP
<img src=""/>
<img src=""/>

colony PCR

F-3,F-4,H-1,H-5,J-2
<img src=""/>
<img src=""/>
OK!

ligation,TF

E-3(D-2 XP+A-1SP)
E-3CP(D-2 XP+A-1CP SP)

preculture

F-3,F-4,H-1,H-5,J-2#1,G-5

Lab member

<p>Yoshikawa,Nakashima

</p>

Contents

TF

E-3(chemical)

miniprep

F-3,F-4,H-1,H-5,J-2(made glycerol stock)
G-5

TF

E-3CP(electroporation)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Takemura

Contents

digestion,gel extraction

A-1CP SP,pSB1C3(A-4) EP,D-2 XP,E-21 XP,G-5 EP,H-5 EP,J-2 SP
<img src=""/>
<img src=""/>

Gel making

ligation

G-5CP(G-5 EP+pSB1C3 EP)
H-5CP(H-5 EP+pSB1C3 EP)
J-5(J-2 SP+E-21 XP)
E-3(A-1CP SP+P-2 XP)

preculture

E-5,E-11,E-22
K-1,E-23',Z-1(for assay)

PCR

VF2-(ligation product of E-3)-VR E-3 lig:1uL
VF2:1uL
VR:1uL
Taq:5uL
MilliQ:2uL
Total:10uL
extention:1m12sec
<img src=""/>
We observed the band which had expected length.
The ligtion must be OK!

Lab member

Yoshikawa.Nakamura,Tsukada,Tara,Yamanaka

Contents

miniprep

E-2,E-5,E-11

digestion,gel extraction

We could not find D-2 sample(probably accidentally disposed).
So we stopped it.

colony PCR

G-5,H-5,J-5
<img src=""/>
H-5,J-5#1,2,3,4:OK!

Making M9 medium

1M MgSO4 50ml
1M CaCl2 10ml
20% glucose 50ml

digestion(cutcheck)

G-5 E,G-5 P,G-5 non-cut
<img src=""/>
Strange bands appeared.
We decided to read a sequence of this part.

Lab member

Nakashima,Tara,Nakamura,Takemura

Contens

miniprep

H-5CP,G-5CP,J-5(made glycerol stock)

sequence order

E-2,E-8,E-9,E-19,F-1,F-3,F-4,G-5,H-1,H-4
H-5(VF-VR,Psigma2F-anti2R),J-2,J-4,J-5,J-6

Lab member

Nakashima,Nakamura,Tara

Contents

assay

completely failed

In M9 medium, Escherichia.coli JM109 did not grow well.(doubling time:1h)
The glycerol stock needed to be put a lot.

culture

F-2,F-3,F-4(for M9 check)

PCR

G-1(F,R),G-4(F,R),G-5(F,R),H-1(F,R),H-4(F,R),H-5(F,R)
Template:1ng/uL,1uL

Making M9 medium

5*M9 24mL
20% Glu 1.2mL
MgSO4 240uL
CaCl2 12uL
Amino acid 10mL
mess up to 1L

PCR check

<img src=""/>

OK!(except G-5)

PCR

G-1,G-4,H-1,H-4
extension time:6m30sec
EpCAM nested
95C 3min-(-95C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C

preculture

E-17,E-18,F-1,F-2,F-3,F-4,Z-1(M9)
E-23'(LB)

Lab member

Nakashima,Tara,Nakamura

Contents

PCR clean up and check

<img src=""/>
G-1,G-4,H-1,H-4:Band in correct position and unexpected position.
EpCAM:No band

digestion,gel extraction

<img src=""/>
<img src=""/>
G-1deg linear EP,G-4deglinear EP,H-1deg linear EP,H-4deg linear EP(A-4)
(deg:degradation tag)

PCR

EpCAM nested
94C 3min-(-94C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C <img src=""/>
No band

gel making

ligation,TF

H-1'(H-1'linear EP+pSB1C3 EP)
H-4'(H-4'linear EP+pSB1C3 EP)
G-1'(G-1'linear EP+pSB1C3 EP)
G-4'(G-4'linear EP+pSB1C3 EP)

PCR

EpCAM nested
95C 3min-(-95C 30sec-46C 30sec-72C 3min-)*30-72C 5min-4C <img src=""/>
No band

Lab member

Yoshikawa

assay1

F-2 in 20mL culture
When OD600=0.516,we put 10% arabinose 20uL.
F-1 in 20mL culture
When OD600=0.514,we put 10% arabinose 20uL.
Z-1 in 20mL culture
When OD600=0.544,we put 10% arabinose 20uL.
O/N culture

Culture in 3mL:disposed(OD600 value did not agrees with each other.)

colony PCR

H-1deg,H-4deg,G-1eg,G-4deg
We confirmed by fluorescence.

preculture

H-1deg,H-4deg#1,G-1eg,G-4deg

Lab member

Yoshikawa

Contents

miniprep

H-1deg,H-4deg#1,G-1eg,G-4deg

preculture

(M9)

E-15CP,E-17,E-18CP,I-2,F-1,2,3,4,Z-1

</p>

Lab member

Yoshikawa,Nakashima,Tara

Contents

assay1,3

E-15,I-2,F-1,F-2,F-3,F-4,F-17,E-18,Z-1
Measured the OD 600 value.
Except I-2,F-2,the value is more than 1.0.
F-1,F-3,E-17,Z-1(20 fold dilution):Culture in 3mL. The composition of M9 proved to be wrong,so disposed.

Making M9 medium

5*M9 200mL
20% Glu 10mL
amino acids 10mL
1M MgSO4 2mL
1M CaCl2 100uL
MilliQ 778mL
Total 1000mL

result of OD600

F-3(non-Chloramphenicol)
1h:0.094
2h:0.086
F-3(Chloramphenicol) 1h:0.074
2h:0.073

assay1

(tentative)

We measured the fluorescence of F-1,F-2,Z-1(culture in 20mL).
Ex:501nm
F-2:We observed a peak in 511nm.
F-1,Z-1:No peak

</p>

preculture

F-1,F-2,F-3,F-4,E-15CP,I-2,Z-1,E-17,E-18CP(M9 medium)
E-23'(LB medium)

Lab member

Yoshikawa,Nakashima,Tara,Nakamura

Contents

assay

F-1,F-13,E-17,Z-1,E-15,I-2
culture in M9
except E-15,OD600 is more than 1.0.
E-15:over 0.85
F-2:only 0.3
E-23':over 2.0,culture in LB medium
All samples was cultured in 3 mL.(n=5)
F-1,Z-1:cultured in 20mL(flask)(n=1)

OD600 before subculture

E-15:1.113
E-11:0.933
E-18:1.190
E-23':forgot to measure
F-1:0.927
F-2:0.374
F-3:0.878
F-4:0.992
I-2:0.888
Z-1:1.084

PCR

J-5(F,R),J-6(F,R)
J-5:OK!
extension time
F:1800+200=2000bp,4min
R:1850+200=2050bp,4min

PCR

J-6(F,R)
J-5R
extension time:1min

Lab member

Yoshikawa,Nakashima,Tara

Contents

making gel

subculture

Escherichia.coliMG1655
100 fold dilution
20mL,flask

PCR clean up and check

<img src=""/>
<img src=""/>
J-6F:low concentration
J-6R:shorter band is OK!
We decided that we made J-5,J-6
as H5deg-positive feedback circuit, and H6deg-positive feedback circuit.

sequence order

G-1deg,G-4deg,H-1deg,H-4deg

TF

(electroporation)

F-1,F-2,F-3,F-4,E-17,E-18CP,Z-1,I-2,E-15
G-1deg,G-4deg,H-1deg,h-4deg,J-4,J-4
also,2mL culture as recovery.

</p>

Lab member

Yoshiakwa,Nakashima,Tara

Contents

digestion,gel extraction

E-19 XP,E-20 XP,H-1deg SP,H-4deg SP

making gel

making plate

Chloramphenicol*10

ligation

J-5deg(E-19XP+H-1degSP)
J-6deg(E-20XP+H-4degSP)
We did not have competent cell of E.coli MG1655, so put it in freezer.

preculture

F-1,E-17,E-15,Z-1,I-2:both in LB medium and M9 medium.
MG1655:LB medium

Lab member

Yoshikawa,Nakashima,Tara,Yamanaka

assay1,3

I-2,E-15,Z-1,F-1,E-17(made glycerol stock,subculture in 20 fold dilution)
MG1655 did not grow, because we accidentally put antibiotics.
OD600(O/N culture)
E-15:1.795
E-17:1.781
F-1:1.937
I-2:1.886
Z-1:1.780

making M9 medium