Team:Chiba/jk/γ

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24,August 2008
TRANSFORMATION
BBa_J63001(yeast optimized YFP)-->colony
Digestion
BBa_F2620(1)①
BBa_F2620(2)②
Menbren Venus YFP③

Sample No	①	②	③
DNA tamplate	3	3	3
Buffer3	1	1	1
dH2O	6	6	6
EcoRⅠ	0.5	0.5	0.5
TOTAL	10	10	10

->37℃ 30min

Sample No	①(afetr digestion)	②(after digestion)	③(afetr digestion)
DNA tamplate	10	10	10
loading Dye	2	2	2
TOTAL	12	12	12

Sample No	①	②	③
DNA tamplate	2	2	2
loading Dye	1	1	1
dH2O	3	3	3
TOTAL	6	6	6

->135V 30min

25,August,2008
PCR
BBa_E2030①
BBa_J33202②
BL21③
BBa_J52008④
BBa_I712019⑤

>

Sample No	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
FW primer	5(Venus_YFP_fwd)	5(LacZ_fwd)	5(LacZ_fwd)	5(rLUC_fwd)	5(fLuc_fwd)
VR primer	5(VR)	5(LacZ_rev)	5(LacZ_rev)	5(VR)	5(VR)
dNTPmix	10	10	10	10	10
thermo pol buffer	10	10	10	10	10
DNA pol(VENT)	1	1	1	1	1
dH2O	68	68	68	68	68
TOTAL	100	100	100	100	100

->Gel

>

Sample No	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
loading Dye	1	1	1	1	1
dH2O	4	4	4	4	4
TOTAL	6	6	6	6	6

MINI prep
Venus
BBa_F2620
TRANSFORMATRION
Venus
PUC19
pGFPUV
pGEX Venus YFP
Membren Venus YFP
PCR
BBa_J63001①

Sample No	①
DNA tamplate	1
FW primer(Venus)	5
VR primer	5
dNTPmix	10
thermo pol buffer	10
DNA pol(VENT)	1
dH2O	68
TOTAL	100

26,August,2008
Gel check
BBa_F2620
BBa_F2620
BBa_J63001

>

Sample No	①	②	③	④	⑤
DNA tamplate	1	1	1	1	1
Loading Dye	1	1	1	1	1
dH2O	4	4	4	4	4
TOTAL	6	6	6	6	6

Digestion
vector:BBa_F2620

</tr>

Sample No	BBa_F2620
DNA tamplate	100
SpeⅠ	2
PstⅠ	2
BSA(x10)	13
Buffer2	13
TOTAL	130

insert:BBa_J52008,BBa_E2030

</tr>

Sample No	J52008	E2030
DNA tamplate	30	30
XbaⅠ	1	1
PstⅠ	1	1
DpnⅠ	1	1
Buffer3	4	4
BSA(x10)	4	4
TOTAL	40	40

->37℃　3hour
->SAP
->37℃ 30min
->65℃ 15min
->Zymo Clean
Ligation
vector:BBa_F2620 insert:BBa_J52008①
vector:BBa_F2620 insert:BBa_E2030②
Negative control:BBa_F2620③

Sample No	①	②	③
vector	5	5	5
inser	10	7.5	0
Ligase Buffer	4	4	4
Ligase	1	1	1
dH2O	0	2.5	10
TOTAL	20	20	20

->RT 2hour
->TRANSFORMATION XL10G
->37℃ over night
->colony PCR
->MINI PREP

28,August,2008
TIME RESPONCE(liquid)

29,August,2008
TIME RESPONCE(solid)

30,August,2008
TRANSFORMATION
mcherry
PCR
BBa_J52008(rLuc)

</tr>

Sample No	BBa_J52008
DNA tamplate	1
rLuc_fwd	2.5
VR	2.5
thermo pol Buffer	5
dNTP	5
Vent pol	0.5
dH2O	34
TOTAL	50

31,August,2008
digestion
BBa_R0010①
PCR product(BBa_J52008)②

Sample No	①	②
DNA	10	10
SpeⅠ	1	0
PstⅠ	1	1
XbaⅠ	0	1
BSA(x100)	0.5	0.5
Buffer2	5	0
Buffer3	0	5
dH20	28.5	28.5
TOTAL	50	50

->37℃ 3hour
->①20μl refine
->②20μl refine

1,September,2008
Ligation
vector:BBa_R0010 insert:BBa_J52008①
negative control:BBa_R0010②

</tr>

Sample No	①	②
vector	2	2
insert	3	0
Ligase Buffer	2	2
Ligase	1	1
dH2O	3	5
TOTAL	10	10

->R/T 2hour

TRANSFORMATION
BBa_R0079(Las promoter)
BBa_R0071(RhlR promoter)
BBa_R0077(cinR promoter+RBS)
BBa_R0078(cnR promoter)
BBa_R0062(LUX promoter)

2,September,2008
TIME RESPONCE (Solid)

COLONY PCR
BBa_R0010+BBa_J52008

</tr>

Sample No	①
culture	1
Fwd primer	1.5
Rev primer	1.5
Thermo pol Buffer	3
dNTP mix	3
Taq DNA pol (NEB)	0.2 (1 unit)
dH2O	19.8
TOTAL	30

MINI Prep
BBa_R0079
BBa_R0071
BBa_R0077
BBa_R0078
BBa_R0062

3,September,2008
Digestion test
BBa_R0079①
BBa_R0071②
BBa_R0077③
BBa_R0078④
BBa_R0062⑤

</tr>

Sample No	①~⑤
DNA tamplate	5
Buffer3	1
dH2O	4
EcoRⅠ	0.1
TOTAL	10

->37℃30min
->Gel cheack

Sample No	①~⑤
DNA(digestion sample)	10
loading Dye	2
TOTAL	12

4,Septempber,2008

5,September,2008
TIME RESPONCE(liquid)
Digestion test
BBa_J52008+BBa_F2620
BBa_E2030+BBa_F2620

Sample No	each
DNA(digestion sample)	10
loading Dye	2
TOTAL	12

Luciferase cheack->no light

7,September,2008
Cloning
Digestion
vector:BBa_R0040(ptet)① 75ng/μl
insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
vector:BBa_R0010(pLac)⑤ 100ng/μl
insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl

Sample No	①	②	③	④	⑤	⑥
DNA(digestion sample)	50	54	54	45	14	54
SpeⅠ	1	0	0	0	1	0
PstⅠ	0.5	0.5	0.5	0.5	0.5	0.5
XbaⅠ	0	0.5	0.5	0.5	0	0.5
BSA(x10)	0.6	7	7	6	2	7
Buffer2	6	-	-	-	2	-
Buffer3	-	7	7	6	-	7
dH2O	1.9	1	1	2	0.5	1
TOTAL	60	70	70	60	20	70

----->37℃ 3hour
----->Gel Extraction

Sample No	①	②	③	④	⑤	⑥
DNA(digestion sample)	6	10	10	10	4	10
Loading Dye	2	2	2	2	1	2
dH2O	4	-	-	-	1	2
TOTAL	12	12	12	12	6	12
well	10	7	7	6	5	7

->DNA purification->①-④,⑥->7μl ⑤->6μl

8,Septmeber,2008
SAP
BBa_R0040(ptet)①
BBa_R0010(pLac)⑤

Sample No	①	⑤
DNA(digestion sample)	7	6
SAP/td>	2	2
SAP Buffer	3	3
TOTAL	30	30
TOTAL	30	30

->37℃ 1hour
->65℃ 30min
->DNA purification
->21μl 6μl
->Gel cheack

Sample No	each
DNA(digestion sample)	1
Loading Dye	1
dH2O	4
TOTAL	6

vector:BBa_R0040(ptet)① 75ng/μl
insert:BBa_S03158(RBS+RhlR)② 33.6ng/μl
insert:BBa_S03156(RBS+lasR)③ 33.6ng/μl
insert:BBa_S03160(RBS+cinR)④ 38.4ng/μl
vector:BBa_R0010(pLac)⑤ 100ng/μl
insert:BBa_J52008(RBS+rLuc)⑥ 33.6ng/μl

</tr>

Sample No	①&②	①&③	①&④	①	⑤&⑥	⑤
DNA(vector)	3	3	3	3	5	2.1
DNA(insert)	3	3	3	-	3.1	-
Ligase	1	1	1	1	1	1
Ligase Buffer(x5)	2	2	2	2	2	2
dH2O	1	1	1	4	8.9	4
TOTAL	10	10	10	10	20	10

9,September,2008

10,September,2008
TIME Responce(liquid)
TIME Responce(solid)
Colony PCR
->Gel cheack
MINI Prep

13,September,2008

14,September,2008
Digestion test
BBa_Q04510
BBa_I716463
BBa_C0051
BBa_C0070
BBa_J06620
BBa_J22141
BBa_C0076
BBa_I716542
BBa_I716462
BBa_C0078
BBa_J01101
BBa_C0071
BBa_I712019
BBa_S01416
BBa_C0079
BBa_C0077
BBa_J23007
BBa_I7100(x2)
BBa_S01416

Sample No	each
DNA(digestion sample)	3
Buffer2(x10)	1
dH2O	6
EcoRⅠ	0.075
TOTAL	10

->37℃ 1hour
Gel cheack

Sample No	each
DNA(digestion sample)	10
Loading Dye	2
TOTAL	12

16,September,2008
Digestion test
BBa_R0040(ptet)+BBa_S03158(RBS+RhlR)(x2)
BBa_R0040(ptet)+BBa_S03156(RBS+lasR)(x5)
BBa_R0040(ptet)+BBa_S03160(RBS+cinR)(x2)
BBa_S03158(RBS+RhlR)
BBa_S03156(RBS+lasR)
BBa_S03160(RBS+cinR)

</tr>

Sample No	each
DNA	3
EcoRⅠ	0.083
PstⅠ	0.083
BSA(x10)	1
Buffer3(x10)	1
dH2O	5
TOTAL	10

->37℃ 1hour
Gel cheack

Sample No	each
DNA	10
Loading Dye	2
TOTAL	12

TRANSFORATION
BBa_J37019(LuxI+RBS+LuxR)->no colony
BBa_J37003(RBS+LuxR)
BBa_J37020(LuxI+LuxR+pLux+GFP)
BBa_J37032(pLux+GFP)
PCR
Venus YFP(ptet+LuxR+YFP)

Sample No	YFP
DNA	1
Venus Fwd	5
Rev (VR)	5
Thermo pol Buffer	10
dNTP	10
Vent	1
dH2O	68
TOTAL	100

17,September,2008
TRANSFORMATION
BBa_I13500(RBS+GFP)
BBa_J37019(Luxpr+LuxR)-->no colony
MINI Prep
BBa_J37033(RBS+LuxR)
BBa_J37020(Luxpr+LuxR+LuxR+GFP)
BBa_J37032(pLux+GFP)
Digestion
BBa_I7100(ptetR+RBS)
BBa_S03119(ptet+LuxR)
Ligation product(ptet+LasR)
Ligation product(ptet+RhlR)
Ligation product(ptet+cinR)

Sample No	each
DNA template	25
EcoRⅠ	1
PstⅠ	1
BSA(x10)	4
EcoRⅠ Buffer	4
dH2O	5
TOTAL	40

-->37℃ 1hour
->Next day

18,September,2008
SAP 1μl
->37℃　1hour
->30min　65℃
Zymo ->25μl
Ligation
vector:BBa_I7100
insert:BBa_S03119(ptet+LuxR),Ligation product(ptet+LasR,ptet+RhlR,ptet+cinR)

Sample No	Vector&insert	Vector
DNA(vector)	3.5	3.5
DNA(insert)	7.5	-
Ligase	1	1
Ligase Buffer	4	4
dH2O	4	4
TOTAL	20	20

TRANSFORMATION
BBa_J54103(RBS+GFP)
BBa_P2006

Gel Cheack
Ligation product(Venus YFP)
BBa_J37033
BBa_J37020
BBa_J37032

Sample No	each
DNA	each
Loading Dye	1
dH2O	4
TOTAL	6

19,September,2008
colony PCR
ptet+LuxR(x16)
ptet+LasR(x7)
ptet+RhlR(x5)
ptet+cinR(x5)

22,September,2008
Time delay

23,September,2008
Digestion test
BBa_E0240

Sample No	E0240
DNA	2
EcoRⅠ	0.5
PstⅠ	0.5
BSA(x10)	1
EcoRⅠ Buffer(x10)	1
dH2O	5
TOTAL	10

Digestion
Vector:BBa_E0240
Insert:BBa_R0062
Vector:BBa_I7100
Insert:BBa_R0062
Insert:BBa_J37033

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