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

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