Contents 1 Golden Gate Assembly Notebook 1.1 August 10 1.2 September 6 1.2.1 PCR and Electrophoresis assay 1.3 September 7 1.3.1 PCR and Electrophoresis assay 1.4 September 10 1.4.1 PCR and Electrophoresis assay 1.4.2 PCR and Electrophoresis assay 1.4.3 PCR and Electrophoresis assay 1.4.4 PCR and Electrophoresis assay 1.4.5 PCR and Electrophoresis assay 1.5 September 12 1.5.1 Golden Gate Assembly 1.6 September 12 1.6.1 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 1.12.3 Transformation

Golden Gate Assembly Notebook

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.

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

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

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

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, 51℃ 30sec)x30cycles, 68℃ 30sec

PCR and Electrophoresis assay

①PCR product of psB1K3 ②psB1K3 ③psB1C3 ④psB1K3 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

PCR and Electrophoresis assay

①DT ②DT ③CFP ④CFP

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 ②DT ③DT

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, 50℃ 30sec)x30cycles, 68℃ 30sec

September 12

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

September 12

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

Transformation