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From 2012.igem.org

Contents 1 1.1 September 6 1.1.1 PCR and Electrophoresis assay 1.2 September 7 1.2.1 PCR and Electrophoresis assay of psB1K3 1.3 September 10 1.3.1 PCR and Electrophoresis assay of CFP 1.3.2 PCR and Electrophoresis assay of lacI 1.4 PCR 1.5 September 11 1.5.1 PCR and Electrophoresis assay 1.5.2 PCR and Electrophoresis assay 1.6 September 12 1.6.1 Purification of PCR product 1.6.2 Golden Gate Assembly 1.6.3 Transformation 1.7 September 13 1.7.1 Transformation 1.8 September 14 1.8.1 Liquid Culture 1.9 September 15 1.9.1 Miniprep 1.9.2 Restriction Enzyme Digestion 1.10 September 17 1.10.1 Electrophoresis assay 1.11 September 18 1.11.1 Restriction Enzyme Digestion 1.11.2 Colony PCR 1.12 September 19 1.12.1 DpnI Restriction Enzyme Digestion of psB1K3 1.12.2 Ligation of psB1K3 1.13 September 20 1.13.1 Transformation of psB1K3 1.14 September 21 1.14.1 PCR of psB3C5 and Electrophoresis assay 1.14.2 PCR and Electrophoresis assay 1.14.3 PCR of DT and Electrophoresis assay 1.15 September 22 1.15.1 Colony PCR (Colony plated in September 20) 1.15.2 PCR of Primers and Electrophoresis assay 1.15.3 Ligation 1.16 September 23 1.16.1 Transformation of product of ligation(9/22) 1.17 September 24 1.17.1 Transformation of product of ligation(9/22) 1.18 September 25 1.18.1 Liquid Culture 1.18.2 Ligation 1.18.3 Transformation of product of ligation(9/25)

September 6

PCR and Electrophoresis assay

①psB1K3 ②lacI ③GFP ④GFP ⑤RFP ⑥RFP ⑦CFP ⑧DT

Quick Taq	primer F	Primer R	DNA(①-⑧)	milliQ	total
12.5	0.5	0.5	1	10.5	25

94℃ 2min, (94℃ 30sec, 50℃ 30sec)x25cycles, 68℃ 50sec

September 7

PCR and Electrophoresis assay of psB1K3

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(psB1K3)	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min

September 10

PCR and Electrophoresis assay of CFP

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(CFP)	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 45sec

PCR and Electrophoresis assay of lacI

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(lacI)	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 30sec

PCR

①PCR product of psB1K3 ②psB1K3 ③psB1C3 ④psB1C3 ⑤PCR product of CFP

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(①-⑤)	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min20sec

September 11

PCR and Electrophoresis assay

①DT ②PCR product of DT ③PCR product of CFP ④CFP(2011 plate)

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(①-④)	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 1min

PCR and Electrophoresis assay

①DT 92.1ng/µl ②DT 28.0ng/µl ③DT

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(①-③)	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 50℃ 30sec)x30cycles, 68℃ 30sec

September 12

Purification of PCR product

psB1K3(9/10④) 55µg/ml lacI(9/10) 90µg/ml GFP(9/6) 42µg/ml RFP(9/6) 71µg/ml DT(9/11) 81µg/ml GFP 37µg/ml RFP 57µg/ml

Golden Gate Assembly

psB1K3	lacI	GFP(2'-3)	RFP(3'-5)	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
1.8	0.1	1.0	0.6	0.1	1.5	0.15	1.0	1.0	8.15	15

psB1K3	lacI	GFP(2'-5)	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
1.8	0.1	1.0	0.1	1.5	0.15	1.0	1.0	8.75	15

psB1K3	lacI	GFP(2'-3)	RFP(3'-5)	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	0.2	1.0	1.0	0.2	1.5	0.15	1.0	1.0	6.95	15

psB1K3	lacI	GFP(2'-3)	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	0.2	1.0	0.2	1.5	0.15	1.0	1.0	7.95	15

Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 13

Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 14

Liquid Culture

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 15

Miniprep

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

①4.0 µg/ml ②17 µg/ml ③15 µg/ml ④8.0 µg/ml

Restriction Enzyme Digestion

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

DNA(①-④)	10x NEB Bufer3	BSA	EcoR1	MilliQ	Total
9	2	0.5	0.5	8	20

September 17

Electrophoresis assay

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 18

Restriction Enzyme Digestion

③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

DNA(③,④)	10x H Bufer	BSA	EcoR1	MilliQ	Total
15	3	0.5	0.5	11	30

Colony PCR

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

2x Quick Tag Dye Mix	VF primer	VR primer	MiliQ	Total
25	1	1	23	50

September 19

DpnI Restriction Enzyme Digestion of psB1K3

psB1K3	DpnI	Total
10	0.2	10.2

37℃ 1h incubate

Ligation of psB1K3

psB1K3	Ligation High	MiliQ	TH Kinase	Total
2	5	7	7	15

16.0℃　1.5h

September 20

Transformation of psB1K3

September 21

PCR of psB3C5 and Electrophoresis assay

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(psB3C5)	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min

PCR and Electrophoresis assay

①psB3C5 ②psB1K3

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(①,②)	KOD plus	milliQ	total
5.0	5.0	2.0	1.5	1.5	1.0	1.0	33	50

94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min)x30cycles

PCR of DT and Electrophoresis assay

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA(DT)	KOD plus	milliQ	total
5.0	5.0	2.0	1.5	1.5	1.0	1.0	33	50

94℃ 2min, (94℃ 15sec, 50℃ 30sec, 68℃ 30sec)x30cycles

September 22

Colony PCR (Colony plated in September 20)

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA	KOD plus	milliQ	total
5.0	5.0	2.0	1.5	1.5	1.0	1.0	33	50

94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min20sec)x25cycles

PCR of Primers and Electrophoresis assay

①J23106(1'-2) ②J23106(2'-3) ③J23106(3'-4) ④J23106(4'-5) ⑤J23106(3'-5) ⑥J23106(2'-5) ⑦J23106(1'-5) ⑧araC(1'-6) ⑨araC(5'-6)

10x Buffer KOD plus	2mM dNTPs	25mM MgSO4	F primer	R primer	DNA	KOD plus	milliQ	total
2.5	2.5	1.0	0.75	0.75	0.5	0.5	16.5	25

94℃ 2min, (94℃ 15sec, 56℃ 30sec)x30cycles, 68℃ 20sec

①J23106(1'-2) 60µg/ml ②J23106(2'-3) 63µg/ml ③J23106(3'-4) 48µg/ml ④J23106(4'-5) 60µg/ml ⑤J23106(3'-5) 62µg/ml ⑥J23106(2'-5) 40µg/ml ⑦J23106(1'-5) 29µg/ml ⑧araC(1'-6) 60µg/ml ⑨araC(5'-6) 62µg/ml

Ligation

4 promoters

psB3C5	①J23106 (1'-2)	②J23106 (2'-3)	③J23106 (3'-4)	④J23106 (4'-5)	⑨araC (5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	2.0	2.0	2.5	2.0	2.0	3.0	3.0	1.5	0.15	1.0	1.0	7.85	30

3 promoters

psB3C5	①J23106 (1'-2)	②J23106 (2'-3)	⑤J23106 (3'-5)	⑨araC (5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	2.0	2.0	2.5	2.0	3.0	3.0	1.5	0.15	1.0	1.0	9.85	30

2 promoters

psB3C5	①J23106(1'-2)	⑥J23106(2'-5)	⑨araC(5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	2.0	2.5	2.0	3.0	3.0	1.5	0.15	1.0	11.85	30

1 promoter

psB3C5	⑦J23106(1'-5)	⑨araC(5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	2.5	2.0	3.0	3.0	1.5	0.15	1.0	1.0	13.85	30

0 promoter

psB3C5	⑨araC(5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	2.0	3.0	3.0	1.5	0.15	1.0	1.0	16.35	30

September 23

Transformation of product of ligation(9/22)

September 24

Transformation of product of ligation(9/22)

September 25

Liquid Culture

colony1,2,3 of plate2,3

Ligation

4 promoters

psB3C5	① J23106 (1'-2)	② J23106 (2'-3)	③ J23106 (3'-4)	④ J23106 (4'-5)	⑨ araC (5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	0.2	0.3	0.2	0.2	0.2	1.0	0.2	1.5	0.15	1.0	1.0	7.05	15

3 promoters

psB3C5	①J23106 (1'-2)	②J23106 (2'-3)	⑤J23106 (3'-5)	⑨araC (5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	0.2	0.3	0.2	0.2	1.0	0.2	1.5	0.15	1.0	1.0	7.25	15

2 promoters

psB3C5	①J23106 (1'-2)	⑥J23106 (2'-5)	⑨araC (5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	0.2	0.3	0.2	1.0	0.2	1.5	0.15	1.0	1.0	7.45	15

1 promoter

psB3C5	⑦J23106 (1'-5)	⑨araC (5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	0.6	0.2	1.0	0.2	1.5	0.15	1.0	1.0	7.35	15

0 promoter

psB3C5	⑨araC (5'-6)	GFP	DT	10x NEB T4 Buffer	100x BSA	BsaI	NEB T4 ligase	milliQ	total
2.0	0.2	1.0	0.2	1.5	0.15	1.0	1.0	7.95	15

(37℃ 3min, 16℃ 4min)x25cycles,

Transformation of product of ligation(9/25)