Team:Kyoto/Notebook

From 2013.igem.org

(Difference between revisions)

Revision as of 02:03, 28 September 2013

1 Calender
2 Aug 8
- 2.1 Transformation
3 Aug 9
- 3.1 Transformation
4 Aug 10
- 4.1 Colony PCR
- 4.2 Electrophoresis
- 4.3 Liquid Culture
- 4.4 LB Medium Plate(+Ampicillin)
- 4.5 Transformation
5 Aug 11
- 5.1 Miniprep
- 5.2 Colony PCR
- 5.3 Restriction Enzyme Digestion
- 5.4 Electrophoresis
6 Aug 15
- 6.1 Liquid Culture
- 6.2 Transformation
- 6.3 Miniprep
7 Aug 16
- 7.1 Colony PCR
- 7.2 Electrophoresis
- 7.3 Master Plate
- 7.4 Transformation
- 7.5 Liquid Culture
8 Aug 17
- 8.1 Colony PCR
- 8.2 Electrophoresis
- 8.3 Liquid Culture
- 8.4 Master Plate
- 8.5 Miniprep
- 8.6 Restriction Enzyme Digestion
- 8.7 Electrophoresis
- 8.8 Miniprep
9 Aug 18
- 9.1 Gel Extraction
- 9.2 Ligation
- 9.3 Restriction Enzyme Digestion
- 9.4 Electrophoresis
- 9.5 Colony PCR
- 9.6 Liquid Culture
- 9.7 LB Medium Plate
- 9.8 Transformation
- 9.9 Electrophoresis
10 Aug 19
- 10.1 Colony PCR
- 10.2 Gel Extraction
- 10.3 LB Medium Plate
- 10.4 Restriction Enzyme Digestion
- 10.5 Ligation
- 10.6 Electrophoresis
- 10.7 Transformation
- 10.8 Gel Extraction
- 10.9 Liquid Culture
- 10.10 Master Plate
11 Aug 20
- 11.1 Miniprep
- 11.2 LB Medium Plate
- 11.3 Ristriction Enzyme Digestion
- 11.4 Transformation
- 11.5 Electrophoresis
12 Aug 21
- 12.1 Plusgrow Medium
- 12.2 Liquid culture
- 12.3 Gel Extraction
- 12.4 Ristriction Enzyme Digestion
- 12.5 Electrophoresis
- 12.6 Gel Extraction
- 12.7 Electrophoresis
- 12.8 Ligation
- 12.9 Transformation
- 12.10 LB Medium Plate
- 12.11 Gel Extraction
- 12.12 Miniprep
13 Aug 22
- 13.1 Liquid culture
- 13.2 Colony PCR
- 13.3 Restriction Enzyme Digestion
- 13.4 Electrophoresis
- 13.5 Miniprep
- 13.6 Electrophoresis
14 Aug 23
- 14.1 Electrophoresis
- 14.2 Restriction Enzyme Digestion
- 14.3 Electrophoresis
- 14.4 Gel Extraction
15 Aug 24
- 15.1 Gel Extraction
- 15.2 Restriction Enzyme Digestion
- 15.3 Master Plate
- 15.4 Electrophoresis
16 Aug 26
- 16.1 Gel Extraction
- 16.2 Ligation
- 16.3 Transformation
17 Aug 27
- 17.1 Restriction Enzyme Digestion
- 17.2 Electrophoresis
- 17.3 Colony PCR
- 17.4 Liquid Culture
- 17.5 Restriction Enzyme Digestion
- 17.6 Electrophoresis
- 17.7 Gel Extraction
- 17.8 Transformation
- 17.9 Liquid culture
- 17.10 Genome PCR
- 17.11 Ligation
18 Aug 28
- 18.1 Electrophoresis
- 18.2 Miniprep
- 18.3 Ligation
- 18.4 Gel Extraction
- 18.5 Colony PCR
- 18.6 Restriction Enzyme Digestion
- 18.7 Electrophoresis
- 18.8 Gel Extraction
- 18.9 Colony PCR
- 18.10 Colony PCR
- 18.11 Liquid Culture
- 18.12 Ligation
- 18.13 Liquid Culture
19 Aug 29
- 19.1 Colony PCR
- 19.2 Electrophoresis
- 19.3 Miniprep
- 19.4 Liquid Culture
- 19.5 Restriction Enzyme Digestion
- 19.6 Transformation
- 19.7 Gel Extraction
20 Aug 30
- 20.1 Gel Extraction
- 20.2 Miniprep
- 20.3 Colony PCR
- 20.4 Ethanol Precipitation
- 20.5 Restriction Enzyme Digestion
- 20.6 Electrophoresis
- 20.7 Liquid Culture
21 Aug 31
- 21.1 Gel Extraction
- 21.2 Ligation
- 21.3 Miniprep
22 Sep 1
- 22.1 Transformation
- 22.2 ligation production PCR
- 22.3 Ligation
- 22.4 Liquid Culture
- 22.5 M9 Medium
- 22.6 Miniprep
- 22.7 Kanamycin applicaton
- 22.8 Plating
- 22.9 Liquid Culture
23 Sep 2
- 23.1 Colony PCR
- 23.2 Electrophoresis
- 23.3 Restriction Enzyme Digestion
- 23.4 Liquid Culture
- 23.5 Master Plate
- 23.6 Electrophoresis
- 23.7 Gel Extraction
- 23.8 Colony PCR
- 23.9 Electrophoresis
- 23.10 Master Plate
- 23.11 Liquid Culture
- 23.12 Transformation
24 Sep 3
- 24.1 Electrophoresis
- 24.2 Miniprep
- 24.3 Liquid Culture
- 24.4 Restriction Enzyme Digestion
- 24.5 PCR
- 24.6 Mutatation PCR
- 24.7 Colony PCR
- 24.8 Electrophoresis
- 24.9 Electrophoresis
- 24.10 Gel Extraction
- 24.11 Liquid Culture
25 Sep 4
- 25.1 Ligation
- 25.2 Transformation
- 25.3 Miniprep
- 25.4 Master Plate
- 25.5 Liquid Culture
- 25.6 PCR
- 25.7 Restriction Enzyme Digestion
- 25.8 Transformation
- 25.9 Electrophoresis
- 25.10 Gel Extraction
- 25.11 Gel Extraction
- 25.12 Master Plate
- 25.13 Liquid Culture
- 25.14 Colony PCR
26 Sep 5
- 26.1 Electrophoresis
- 26.2 Miniprep
- 26.3 Ligation
- 26.4 Electrophoresis
- 26.5 Gel Extraction
- 26.6 Colony PCR
- 26.7 Liquid Culture
- 26.8 Transformation
- 26.9 Restriction Enzyme Digestion
- 26.10 Electrophoresis
- 26.11 5x M9 Medium (+EDTA)
- 26.12 Gel Extraction
- 26.13 DNA Purification
- 26.14 Miniprep
- 26.15 Colony PCR
- 26.16 Liquid Culture
27 Sep 6
- 27.1 Electrophoresis
- 27.2 Colony PCR
- 27.3 Liquid Culture
- 27.4 Electrophoresis
- 27.5 Miniprep
- 27.6 Restriction Enzyme Digestion
- 27.7 Electrophoresis
- 27.8 Gel Extraction
- 27.9 Liquid Culture
- 27.10 Colony PCR
28 Sep 7
- 28.1 Miniprep
- 28.2 Electrophoresis
- 28.3 Colony PCR
- 28.4 Ligation
- 28.5 Electrophoresis
- 28.6 Transformation
- 28.7 Restriction Enzyme Digestion
- 28.8 Electrophoresis
- 28.9 Liquid Culture
29 Sep 8
- 29.1 Gel Extraction
- 29.2 Miniprep
- 29.3 Ligation
- 29.4 Restriction Enzyme Digestion
- 29.5 Transformation
- 29.6 Electrophoresis
- 29.7 Gel Extraction
- 29.8 Ligation
- 29.9 PCR
- 29.10 Gel Extraction
30 Sep 9
- 30.1 PCR
- 30.2 Gel Extraction
- 30.3 Miniprep
- 30.4 Restriction Enzyme Digestion
- 30.5 Transformation
- 30.6 Ligation
- 30.7 M9 medium
- 30.8 Colony PCR
- 30.9 Gel Extraction
- 30.10 Ligation
- 30.11 PCR
- 30.12 Liquid Culture
- 30.13 Restriction Enzyme Digestion
- 30.14 PCR
- 30.15 Liquid Culture
- 30.16 Gel Extraction
- 30.17 Electrophoresis
31 Sep 10
- 31.1 Ligation
- 31.2 Miniprep
- 31.3 Restriction Enzyme Digestion
- 31.4 Electrophoresis
- 31.5 Colony PCR
- 31.6 Electrophoresis
- 31.7 Gel Extraction
- 31.8 Liquid Culture
- 31.9 Gel Extraction
- 31.10 Liquid Culture
- 31.11 Colony PCR
- 31.12 Electrophoresis
- 31.13 Transformation
- 31.14 Electrophoresis
32 Sep 11
- 32.1 Electrophoresis
- 32.2 PCR
- 32.3 Electrophoresis
- 32.4 colony PCR
- 32.5 Electrophoresis
- 32.6 colony PCR
- 32.7 Gel Extraction
- 32.8 Ligation
- 32.9 Transformation
- 32.10 Electrophoresis
- 32.11 Restriction Enzyme Digestion
- 32.12 Liquid Culture
- 32.13 PCR
- 32.14 Liquid Culture
33 Sep 12
- 33.1 Miniprep
- 33.2 Electrophoresis
- 33.3 Liquid Culture
- 33.4 Electrophoresis
- 33.5 Gel Extraction
- 33.6 Gel Extraction
- 33.7 Electrophoresis
- 33.8 Colony PCR
- 33.9 Restriction Enzyme Digestion
- 33.10 Colony PCR
- 33.11 Transformation
- 33.12 Electrophoresis
- 33.13 Electrophoresis
- 33.14 Liquid Culture
- 33.15 Colony PCR
- 33.16 Electrophoresis
- 33.17 Restriction Enzyme Digestion
- 33.18 Electrophoresis
  - 33.18.1 Observation through a confocal microscope
- 33.19 Liquid Culture
34 Sep 13
- 34.1 Miniprep
- 34.2 Gel Extraction
- 34.3 Restriction Enzyme Digestion
- 34.4 M9 (EDTA)
- 34.5 Gel Extraction
- 34.6 Liquid Culture
- 34.7 Restriction Enzyme Digestion
- 34.8 Electrophoresis
- 34.9 Ligation
- 34.10 PCR
- 34.11 PCR
- 34.12 Gel Extraction
- 34.13 Transformation
35 Sep 14
- 35.1 Restriction Enzyme Digestion
- 35.2 Liquid Culture
- 35.3 Electrophoresis
- 35.4 Sequence PCR
- 35.5 Gel Extraction
- 35.6 Restriction Enzyme Digestion
- 35.7 Ligation
- 35.8 Colony PCR
- 35.9 Electrophoresis
- 35.10 Liquid Culture
- 35.11 Transformation
- 35.12 Sequence PCR
36 Sep 15
- 36.1 Miniprep
- 36.2 Liquid Culture
- 36.3 Ligation
- 36.4 Restriction Enzyme Digestion
- 36.5 Electrophoresis
- 36.6 Gel Extraction
- 36.7 Ligation
- 36.8 Restriction Enzyme Digestion
- 36.9 Liquid Culture
37 Sep 16
- 37.1 Liquid Culture
- 37.2 Colony PCR
- 37.3 Gel Extraction
- 37.4 Miniprep
- 37.5 PCR
- 37.6 Electrophoresis
- 37.7 Restriction Enzyme Digestion
- 37.8 Ligation
- 37.9 Transformation
- 37.10 Miniprep
- 37.11 PCR
38 Sep 17
- 38.1 Liquid Culture
- 38.2 PCR
- 38.3 Electrophoresis
- 38.4 Ligation
- 38.5 Gel Extraction
- 38.6 Mutation PCR
39 Sep 18
- 39.1 Restriction Enzyme Digestion
- 39.2 Electrophoresis
- 39.3 Electrophoresis
- 39.4 Gel Extraction
40 Sep 19
41 Sep 20
- 41.1 Ligation
- 41.2 Transformation
- 41.3 PCR
- 41.4 Electrophoresis
42 Sep 21
- 42.1 Electrophoresis
- 42.2 Liquid Culture
- 42.3 Master Plate
- 42.4 Restriction Enzyme Digestion
- 42.5 Electrophoresis
- 42.6 PCR
- 42.7 Gel Extraction
43 Sep 22
- 43.1 Colony PCR
- 43.2 Electrophoresis
- 43.3 Miniprep
- 43.4 Colony PCR
- 43.5 Gel Extraction
- 43.6 Restriction Enzyme Digestion
- 43.7 Gel Extraction
- 43.8 Colony PCR
- 43.9 Colony PCR
- 43.10 Ligation
- 43.11 Liquid Culture
- 43.12 digestion
- 43.13 Gel Extraction
- 43.14 Transformation
- 43.15 PCR
- 43.16 Electrophoresis
44 Sep 23
- 44.1 Transformation
- 44.2 Gel Extraction
- 44.3 PCR
- 44.4 Ligation
- 44.5 PCR
- 44.6 Miniprep
- 44.7 Electrophoresis
- 44.8 Gel Extraction
- 44.9 Electrophoresis
- 44.10 Transformation
- 44.11 Ligation
- 44.12 Colony PCR
45 Sep 24
- 45.1 Electrophoresis
- 45.2 Transformation
- 45.3 Liquid Culture
- 45.4 Restriction Enzyme Digestion(PCR product)
- 45.5 Column Refining
- 45.6 Transformation
- 45.7 Liquid Culture
- 45.8 Restriction Enzyme Digestion
- 45.9 Electrophoresis
- 45.10 Miniprep
- 45.11 Gel Extraction
- 45.12 Colony PCR
- 45.13 Restriction Enzyme Digestion
- 45.14 Ligation
- 45.15 colony PCR
- 45.16 Miniprep
- 45.17 Electrophoresis
- 45.18 Gel Extraction
- 45.19 Electrophoresis
- 45.20 Ligation
- 45.21 PCR
46 Sep 25
- 46.1 Electrophoresis
- 46.2 Colony PCR
- 46.3 Electrophoresis
- 46.4 Transformation
- 46.5 Ligation
- 46.6 Restriction Enzyme Digestion
- 46.7 Liquid Culture
- 46.8 Gel Extraction
- 46.9 Electrophoresis
- 46.10 Gel Extraction
- 46.11 Liquid Culture
- 46.12 Ligation
- 46.13 PCR
- 46.14 Transformation
- 46.15 Electrophoresis
- 46.16 Column Refining
- 46.17 Restriction Enzyme Digestion
- 46.18 Liquid Culture
- 46.19 Plating
- 46.20 Colony PCR
- 46.21 RNA Extraction
- 46.22 cDNA　Synthesis
- 46.23 Electrophoresis
- 46.24 Ligation
47 Sep 26
- 47.1 Electrophoresis
- 47.2 Ligasion(Golden Gate Assenbly)
- 47.3 Liquid Culture
- 47.4 BSAI Digestion
- 47.5 Colony PCR
- 47.6 Electrophoresis
- 47.7 Electrophoresis
- 47.8 Liquid Culture
- 47.9 Colony PCR
- 47.10 Ligasion(Golden Gate Assenbly)
- 47.11 RNA Extraction
- 47.12 Liquid Culture
- 47.13 Colony PCR
- 47.14 Electrophoresis
- 47.15 Plating
- 47.16 Restriction Enzyme Digestion
- 47.17 Electrophoresis
48 Sep 27
- 48.1 Gel Extraction
- 48.2 Gel Extraction
- 48.3 Electrophoresis
- 48.4 Liquid Culture
- 48.5 Ligation
- 48.6 Liquid Culture
- 48.7 RNA Extraction
- 48.8 Electrophoresis
- 48.9 Miniprep
- 48.10 Transformation
- 48.11 Miniprep
- 48.12 Restriction Enzyme Digestion
- 48.13 RNA Extraction
- 48.14 DNA sythesis
- 48.15 Electrophoresis
- 48.16 Liquid Culture
- 48.17 RT PCR
- 48.18 Electrophoresis
- 48.19 Digesting cut check
- 48.20 Gel Extraction
- 48.21 RNA Extraction
- 48.22 cDNA Synthesis
49 Sep 28
- 49.1 Electrophoresis
- 49.2 Colony PCR
- 49.3 Electrophoresis

Calender

August
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

September
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30

Aug 8

Transformation

Inoue and Nakamoto

Name	Sample	Competent Cells	Total	Plate
BBa_J44000	2 µL	20 µL (7/30)	22 µL	LB (+Amp)
BBa_J44000	2 µL	20 µL (XL10-gold)	22 µL	LB (+Amp)
BBa_K747028	2 µL	20 µL (XL10-gold)	22 µL	LB (+CP)
BBa_K747028	2 µL	20 µL (7/30)	22 µL	LB (+CP)
BBa_J23100	2 µL	20 µL (XL10-gold)	22 µL	LB (+Amp)
BBa_J23106	2 µL	20 µL (XL10-gold)	22 µL	LB (+Amp)
BBa_E2030	2 µL	20 µL (XL10-gold)	22 µL	LB (+Kan)

Aug 9

Transformation

Kojima and Nakamoto

Name	Sample	Competent Cells	Total	Plate
BBa_J44000	2 µL	20 µL	22 µL	LB (+Amp)
BBa_K747028	2 µL	20 µL	22 µL	LB (+CP)
BBa_J23100	2 µL	20 µL	22 µL	LB (+Amp)
BBa_J23106	2 µL	20 µL	22 µL	LB (+Amp)
BBa_E2030	2 µL	20 µL	22 µL	LB (+Kan)

Aug 10

Colony PCR

Nakamoto

Sample
8/9 BBa_J44000 -(1)
8/9 BBa_J44000 -(2)
NC

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 sec	30 sec	30 sec	30 cycles

Sample
8/9 BBa_J23106
8/9 BBa_J23100 -(1)
8/9 BBa_J23100 -(2)
NC

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 sec	30 sec	1 min	30 cycles

Electrophoresis

Tatsui and Nakamoto

Lane	Sample
	100bp ladder
4	8/9 BBa_J44000 -1
5	8/9 BBa_J44000 -2
6	NC

Lane	Sample
	1kbp ladder
1	8/9 BBa_J23106
2	8/9 BBa_J23100 -1
3	8/9 BBa_J23100 -2
4	NC

Liquid Culture

Nakamoto

Sample	medium
8/9 BBa-J23106	LB(+Amp)
8/9 BBa-J23100-1	LB(+Amp)
8/9 BBa-J23100-2	LB(+Amp)
8/9 BBa-J44000-1	LB(+Amp)
8/9 BBa-J44000-2	LB(+Amp)

LB Medium Plate(+Ampicillin)

Nakamoto & Tatsui

volume	200ml
Bacto T2ypton	2g
Bacto yeast extract	1g
NaCl	1g
0.4M NaOHaq	500µL
Agarose Pouder	2g
Ampicillin	40µL

Transformation

Hirano

Name	Sample	Competent Cells	Total	Plate
tetR	3µL	19µL	22µL	Kan
RBS	3µL	19µL	22µL	Amp
lacZα	3µL	19µL	22µL	CP
lacI	3µL	19µL	22µL	CP
Plac	3µL	19µL	22µL	CP

Aug 11

Miniprep

Nakamoto

DNA	concentration[µg/mL]
8/9 BBa_J44000 -(2)	100
8/9 BBa_J23106	154
8/9 BBa_J23100	144
8/10 T7-His-FT	92

Colony PCR

Kojima and Yasuda

Sample
BBa_B0034 -(2)
BBa_B0034 -(1)
BBa_R0011 -(2)
BBa_R0011 -(1)
NC

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 s	30 s	20 s	30 cycles

Sample
BBa_D0412 -(1)
BBa_D0412 -(2)
NC

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 s	30 s	1 min 30 s	30 cycles

Restriction Enzyme Digestion

Kojima anda Yasuda

8/9 BBa_J44000 2	EcoRI	PstI	10x buffer H	MilliQ	total
2	0.2	0.2	1	6.6	10
2	0.2	--	1	6.8	10
2	--	0.2	1	6.8	10
2	--	--	1	7.0	10

8/9 BBa_J23106	EcoRI	PstI	10x buffer H	MilliQ	total
1.3	0.2	0.2	1	7.3	10
1.3	0.2	--	1	7.5	10
1.3	--	0.2	1	7.5	10
1.3	--	--	1	7.7	10

8/9 BBa_J23100 2	EcoRI	PstI	10x buffer H	MilliQ	total
1.3	0.2	0.2	1	7.3	10
1.3	0.2	--	1	7.5	10
1.3	--	0.2	1	7.5	10
1.3	--	--	1	7.7	10

incubated at 37 °C for 1 hour.

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	8/9 BBa_J44000	EcoRI	PstI
3	8/9 BBa_J44000	EcoRI	--
4	8/9 BBa_J44000	--	PstI
5	8/9 BBa_J44000	--	--
6	8/9 BBa_J23106	EcoRI	PstI
7	8/9 BBa_J23106	EcoRI	--
8	8/9 BBa_J23106	--	PstI
9	8/9 BBa_J23106	--	--
10	8/9 BBa_J23100	EcoRI	PstI
11	8/9 BBa_J23100	EcoRI	--
12	8/9 BBa_J23100	--	PstI
13	8/9 BBa_J23100	--	--

Lane	Sample
1	100bp ladder
2	BBa_B0034 (1)
3	BBa_B0034 (2)
4	BBa_R0011 (1)
5	BBa_R0011 (2)
6	NC
7	100bp ladder

Lane	Sample
1	1kb ladder
2	BBa_P0412 (1)
3	BBa_P0412 (2)
4	NC

Aug 15

Liquid Culture

Nakamoto

Sample	medium
8/10 BBa_B0034(1)	LB (+Amp)
8/10 BBa_B0034(2)	LB (+Amp)
8/10 BBa_R0011(1)	LB (+CP)
8/10 BBa_R0011(2)	LB (+CP)
8/10 BBa_P0412(1)	LB (+CP)
8/10 BBa_P0412(2)	LB (+CP)

Transformation

Nakamoto

Name	Sample	Competent Cells	Total	Plate
BBa_I0500(Pbad/araC)	2	17	19	LB (+Kan)
BBa_F1610(RBS-luxI-DT)	2	17	19	LB (+Amp)
BBa_R0061(Plux)	2	17	19	LB (+CP)
BBa_I13504(RBS-GFP-DT)	2	17	19	LB (+CP)
BBa_I0462(RBS-luxR-DT)	2	17	19	LB (+Amp)
BBa_K117000(lysis1)	2	17	19	LB (+CP)
BBa_K112806(lysis2)	2	17	19	LB (+CP)
BBa_K748002(lysis3)	2	17	19	LB (+CP)
BBa_B0015(DT)	2	17	19	LB (+CP)
BBa_Q04400(RBS-tetR-DT-Ptet)	2	17	19	LB (+Kan)
BBa_I732020(RBS-lacZα-DT)	2	17	19	LB (+CP)
BBa_J23100	2	17	19	LB (+Amp)

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
BBa_B0034(1)	84.1	2.11	2.01
BBa_B0034(2)	86.9	2.03	2.02
BBa_R0011(1)	78.3	2.26	2.02
BBa_R0011(2)	88.9	2.24	2.02
BBa_P0412(1)	15.61	2.10	1.93
BBa_P0412(2)	12.93	2.18	1.91

Aug 16

Colony PCR

Tatsui and Ashida

Sample	base pair
8/15 BBa_I13504(RBS-GFP-DT)(1)	1174
8/15 BBa_I13504(RBS-GFP-DT)(2)	1174
8/15 BBa_I0462(RBS-luxR-DT)(1)	1174
8/15 BBa_I0462(RBS-luxR-DT)(2)	1174
8/15 BBa_K748002(lysis3)	1053
NC	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Sample	base pair
8/15 BBa_R0061(Plux)	344
8/15 BBa_K117000(lysis1)(1)	456
8/15 BBa_K117000(lysis1)(2)	456
8/15 BBa_B0015(DT)	443
8/15 BBa_I732020(RBS-lacZα-DT)	703
NC	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	40s	30cycles

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	BBa_I13504(RBS-GFP-DT) -(1)
3	BBa_I13504(RBS-GFP-DT) -(2)
4	BBa_I0462(RBS-luxR-DT) -(1)
5	BBa_I0462(RBS-luxR-DT) -(2)
6	BBa_K748002(lysis3)
7	NC
8	100bp ladder

Lane	Sample
1	100bp ladder
2	BBa_R0061(Plux)
3	BBa_K117000(lysis1) -(1)
4	BBa_K117000(lysis1) -(1)
5	BBa_B0015(DT)
6	BBa_I732020(RBS-lacZα-DT)
7	NC
8	100bp ladder

Master Plate

Tatsui

Number	Use LB plate (+CP)
1	BBa_I13504(RBS-GFP-DT)(1)
2	BBa_I13504(RBS-GFP-DT)(2)
3	BBa_K748002(lysis3)
4	BBa_R0061(Plux)
5	BBa_K117000(lysis1)(1)
6	BBa_K117000(lysis1)(2)
7	BBa_B0015(DT)
8	BBa_I732020(RBS-lacZα-DT)

Number	Use LB plate (+Amp)
1	BBa_I0462(RBS-luxR-DT)(1)
2	BBa_I0462(RBS-luxR-DT)(2)

Transformation

Hirano

Name	Sample	Competent Cells	Total	Plate
pBR322	1µL	100µL(XL-10gold)	101µL	Amp
pBR322	1µL	50µL(takara)	51µL	Amp
Pbad/araC	2µL	20µL	22µL	Kan
RBS-luxI-DT	2µL	20µL	22µL	Kan

Liquid Culture

Yoshdia

Sample	medium
8/15 BBa_I13504(RBS-GFP-DT))(1)	Plusgrow medium (+CP)
8/15 BBa_I13504(RBS-GFP-DT)(2)	Plusgrow medium (+CP)
8/15 BBa_I0462(RBS-luxR-DT)(2)	Plusgrow medium (+Amp)
8/15 BBa_K748002(lysis3)	Plusgrow medium (+CP)
8/15 BBa_R0061(Plux)	Plusgrow medium (+CP)
8/15 BBa_K117000(lysis1)(1)	Plusgrow medium (+CP)
8/15 BBa_K117000(lysis1)(2)	Plusgrow medium (+CP)
8/15 BBa_B0015(DT)	Plusgrow medium (+CP)
8/15 BBa_I732020(RBS-lacZα-DT)	Plusgrow medium (+CP)

Aug 17

Colony PCR

Nakamoto

Sample	base pair
8/15 BBa_K112806(lysis2) -(1)	828
NC	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Sample	base pair
8/10 BBa_Q0400 -(1)	3995
NC	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

Electrophoresis

No name

Lane	Sample
1	1kbp ladder
2	K112806(lysis2) -1
3	NC
4	Q04400(tetR) -1
5	NC
6	1kbp ladder

Liquid Culture

Nakamoto

Sample	medium
8/15 BBa_K112806(lysis2) -(1)	Plusgrow medium(+CP)
8/15 BBa_Q04400(tetR) -(1)	Plusgrow medium(+Amp)
8/16 pBR322 -(1)	Plusgrow medium(+Amp)

Master Plate

Nakamoto

Number	Use LB plate (+CP)
1	BBa_K112806(lysis2) -(1)

Number	Use LB plate (+Kana)
1	BBa_Q04400(tetR) -(1)

Miniprep

Nakamoto and Murakami

DNA	concentration[µg/mL]	260/280	260/230
K748002(lysis3) -(1)	330	1.66	2.08
B0015(DT) -(1)	188	1.73	1.82
R0061(Plux) -(1)	76	1.64	1.30
K117000(lysis1) -(1)	286	1.71	2.01
K117000(lysis1) -(2)	248	1.69	1.78
I732020(RBS-lacZα-DT) -(1)	198	1.71	1.93
I13504(RBS-GFP-DT) -(1)	248	1.69	2.03
I13504(RBS-GFP-DT) -(2)	246	1.55	1.73
I0462(RBS-luxR-DT) -(1)	326	1.65	1.74

Restriction Enzyme Digestion

Murata, Murakami, Nakamoto, Ashida, Matsumoto and Yoshida

	8/11 J23100 -(2)	SpeI	PstI	Buffer	MilliQ	total
2 cuts	4.0µL	1.0µL	1.0µL	3.0µL	21µL	30µL
1 cut	1.3µL	0.2µL	--	1.0µL	7.5µL	10µL
1 cut	1.3µL	--	0.2µL	1.0µL	7.5µL	10µL
NC	1.3µL	--	--	1.0µL	7.7µL	10µL

	8/15 B0034(RBS) -(2)	SpeI	PstI	Buffer	MilliQ	total
2 cuts	6.0µL	1.0µL	1.0µL	3.0µL	19µL	30µL
1 cut	2.0µL	0.2µL	--	1.0µL	6.8µL	10µL
1 cut	2.0µL	--	0.2µL	1.0µL	6.8µL	10µL
NC	2.0µL	--	--	1.0µL	7.0µL	10µL

	8/17 K117000(lysis1) -(1)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	2.0µL	1.0µL	1.0µL	3.0µL	3.0µL	20µL	30µL
1 cut	1.0µL	0.2µL	--	1.0µL	1.0µL	6.8µL	10µL
1 cut	1.0µL	--	0.2µL	1.0µL	1.0µL	6.8µL	10µL
NC	1.0µL	--	--	1.0µL	1.0µL	7.0µL	10µL

	8/17 K748002(lysis3) -(1)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	2.0µL	1.0µL	1.0µL	3.0µL	3.0µL	20µL	30µL
1 cut	1.0µL	0.2µL	--	1.0µL	1.0µL	6.8µL	10µL
1 cut	1.0µL	--	0.2µL	1.0µL	1.0µL	6.8µL	10µL
NC	1.0µL	--	--	1.0µL	1.0µL	7.0µL	10µL

	8/17 I13504(RBS-GFP-DT) -(1)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	2.0µL	1.0µL	1.0µL	3.0µL	3.0µL	20µL	30µL
1 cut	1.0µL	0.2µL	--	1.0µL	1.0µL	6.8µL	10µL
1 cut	1.0µL	--	0.2µL	1.0µL	1.0µL	6.8µL	10µL
NC	1.0µL	--	--	1.0µL	1.0µL	7.0µL	10µL

	8/17 I0462(RBS-luxR-DT) -(1)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	2.0µL	1.0µL	1.0µL	3.0µL	3.0µL	20µL	30µL
1 cut	1.0µL	0.2µL	--	1.0µL	1.0µL	6.8µL	10µL
1 cut	1.0µL	--	0.2µL	1.0µL	1.0µL	6.8µL	10µL
NC	1.0µL	--	--	1.0µL	1.0µL	7.0µL	10µL

	8/17 I732020(RBS-lacZα-DT) -(1)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	2.0µL	1.0µL	1.0µL	3.0µL	3.0µL	20µL	30µL
1 cut	1.0µL	0.2µL	--	1.0µL	1.0µL	6.8µL	10µL
1 cut	1.0µL	--	0.2µL	1.0µL	1.0µL	6.8µL	10µL
NC	1.0µL	--	--	1.0µL	1.0µL	7.0µL	10µL

	8/11 R6061 -(1)	SpeI	PstI	Buffer	MilliQ	total
2 cuts	8.0µL	1.0µL	1.0µL	3.0µL	17µL	30µL
1 cut	3.0µL	0.2µL	--	1.0µL	5.8µL	10µL
1 cut	3.0µL	--	0.2µL	1.0µL	5.8µL	10µL
NC	3.0µL	--	--	1.0µL	6.0µL	10µL

incubate at 37 °C, for 1 h

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	J23100 -②	SpeI	PstI
2	J23100 -②	SpeI	--
3	J23100 -②	--	PstI
4	J23100 -②	--	--
5	B0034(RBS) -(2)	SpeI	PstI
6	B0034(RBS) -(2)	SpeI	--
7	B0034(RBS) -(2)	--	PstI
8	B0034(RBS) -(2)	--	--
9	1kb ladder	--	--
10	K11700(lysis1) -(1)	XbaI	PstI
11	K11700(lysis1) -(1)	XbaI	--
12	K11700(lysis1) -(1)	--	PstI
13	K11700(lysis1) -(1)	--	--
14	K748002(lysis3) -(1)	XbaI	PstI
15	K748002(lysis3) -(1)	XbaI	--
16	K748002(lysis3) -(1)	--	PstI
17	K748002(lysis3) -(1)	--	--

Lane	Sample	Enzyme1	Enzyme2
18	I13504(RBS-GFP-DT) -(1)	XbaI	PstI
19	I13504(RBS-GFP-DT) -(1)	XbaI	--
20	I13504(RBS-GFP-DT) -(1)	--	PstI
21	I13504(RBS-GFP-DT) -(1)	--	--
22	I0462(RBS-luxR-DT) -(1)	XbaI	PstI
23	I0462(RBS-luxR-DT) -(1)	XbaI	--
24	I0462(RBS-luxR-DT) -(1)	--	PstI
25	I0462(RBS-luxR-DT) -(1)	--	--
26	1kb ladder	--	--
27	I732020(RBS-lacZα-DT) -(1)	XbaI	PstI
28	I732020(RBS-lacZα-DT) -(1)	XbaI	--
29	I732020(RBS-lacZα-DT) -(1)	--	PstI
30	I732020(RBS-lacZα-DT) -(1)	--	--
31	R0061(Plux) -(1)	SpeI	PstI
32	R0061(Plux) -(1)	SpeI	--
33	R0061(Plux) -(1)	--	PstI
34	R0061(Plux) -(1)	--	--

Miniprep

Murata

DNA	concentration[µg/mL]	260/280	260/230
8/17 PBR322(TaKaRa) -(1)	47	1.68	1.55
8/17 K112806(lysis2) -(1)	64	1.67	1.70

Aug 18

Gel Extraction

Yoshida and Kojima

Lane	DNA	Enzyme
1	100kb ladder	--
2	8/11 J23100 -②	--
3	8/11 J23100 -②	--
4	--	--
5	8/17 K117000(lysis1) -1	--
6	8/17 K117000(lysis1) -1	--
7	--	--
8	8/17 K748002(lysis3) -1	--
9	8/17 K748002(lysis3) -1	--
10	--	--
11	8/17 I13504(RBS-GFP-DT) -1	--
12	8/17 I13504(RBS-GFP-DT) -1	--

Lane	DNA	Enzyme
13	1kb ladder	--
14	8/17 I0462(RBS-luxR-DT) -1	--
15	8/17 I0462(RBS-luxR-DT) -1	--
16	--	--
17	8/17 I732020(RBS-lacZα-DT) -1	--
18	8/17 I732020(RBS-lacZα-DT) -1	--

Name	concentration[µg/mL]	260/280	260/230
8/14 J231000	1.7	1.61	0.04
8/17 K748002(lysis3) -1	5.4	2.39	0.45
8/17 I13504(RBS-GFP-DT) -1	3.4	3.33	0.19
8/17 I0462(RBS-luxR-DT) -1	4.4	2.07	0.37
8/17 I732020(RBS-lacZα-DT) -1	2.9	2.17	0.29

Ligation

Ashida

state	Vector		Inserter		Ligation High ver.2
experiment	J23100	9.0	RBS-lacZα-DT	8.6	3.5
experiment	J23100	9.0	RBS-GFP-DT	7.3	3.5
experiment	RBS	1	lysis3	4.6	2.8
experiment	Plux	1	GFP	7.3	3.5
experiment	J23100	9.0	--	--	3.5
experiment	RBS	1.0	--	--	0.5
experiment	Plux	1.0	--	--	0.5

incubate 16°C 1hour

Restriction Enzyme Digestion

Murata and Okazaki

	8/17K112806(lysis2)-1	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	2.0µL	1.0µL	1.0µL	3.0µL	3.0µL	3.0µL	30 µL
1 cuts	1.0&mic6/8ro;L	0.2µL	--	1.0µL	1.0µL	6.8µL	10 µL
1 cuts	1.0µL	--	0.2µL	1.0µL	1.0µL	6.8µL	10 µL

Electrophoresis

Okazaki and Honda

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	lysis1	XbaI	PstI
3	lysis1	XbaI	--
4	lysis1	--	PstI
5	lysis1	--	--
6	lysis2	XbaI	PstI
7	lysis2	XbaI	--
8	lysis2	--	PstI
9	lysis2	--	--
10	100bp ladder	--	--

Colony PCR

No name

Sample	base pair
8/8 LBKana	--
8/16 Pbad araC	--
NC	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30

Liquid Culture

No name

Sample	medium
7/8 LB Kana	Plusgrow(+Kana)
8/16 Pbad araC	Plusgrow(+Kana)

LB Medium Plate

No name

volume	200ml
Bacto Trypton	2g
Bacto yeasy extract	1g
NaCl	1g
Agar Powder	2g

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
8/18 J23100+RBS-LacZα-DT	2µL	20µL	22µL	Amp
8/18 J23100+RBS-GFP-DT	2µL	20µL	22µL	Amp
8/18 J23100+RBS-luxR-DT	2µL	20µL	22µL	Amp
RBS+lysis3	2µL	20µL	22µL	Amp
Plux+GFP	2µL	20µL	22µL	CP
J23100	2µL	20µL	22µL	Amp
RBS	2µL	20µL	22µL	Amp
Plux	2µL	20µL	22µL	CP

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	8/16Pbad araC-1	--	--
3	8/16Pbad araC-2	--	--
4	NC	--	--

Aug 19

Colony PCR

Kojima

Sample	base pair
8/18 J23100 control -(1)	1142
8/18 J23100-RBS-GFP-DT -(1)	1156
8/18 J23100-RBS-GFP-DT -(2)	1156
8/18 J23100-RBS-luxR-DT -(1)	1271
8/18 J23100-RBS-luxR-DT -(2)	1271
8/18 J23100-RBS-lacZα-DT -(1)	670
8/18 J23100-RBS-lacZα-DT -(2)	670
8/18 RBS-lysis3 -(1)	997
8/18 RBS-lysis3 -(2)	997
8/18 P0440(RBS-tetR-DT) -(1)	1154
8/18 P0440(RBS-tetR-DT) -(2)	1154
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Lane	DNA
1	100bp ladder
2	8/18 NC J23100(1)
3	8/18 J23100-RBS-GFP-DT-(1)
4	8/18 J23100-RBS-GFP-DT-(2)
5	8/18 J23100-RBS-luxR-DT-(1)
6	8/18 J23100-RBS-luxR-DT-(2)
7	8/18 J23100-RBS-lacZα-DT-(1)
8	8/18 J23100-RBS-lacZα-DT-(2)
9	8/18 RBS-lysis3-(1)
10	8/18 RBS-lysis3-(2)
11	8/18 R0400(RBS-tetR-DT)-(1)
12	8/18 R0400(RBS-tetR-DT)-(2)
13	NC
14	100bp ladder

Name	concentration[µg/mL]	260/280	260/230
8/19 lysis1 ① (XbaI & PstI)	87.8	1.06	0.82

Sample	base pair
8/18 RBS control -(1)	--
8/18 R0040(Ptet) -(1)	686
8/18 R0040(Ptet) -(2)	686
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

Gel Extraction

Ashida

Lane	DNA	Enzyme
1	100bp ladder	--
3	8/18 K117000(lysis2) -1	XbaI & PstI
5	8/18 K117000(lysis2) -1	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
8/18 lysis2-1(XbaI & PstI)	3.1	3.64	0.19
8/18 lysis2-1(XbaI & PstI)	3.4	1.89	0.13

LB Medium Plate

Nakamoto

volume	200ml
Bacto T2ypton	2g
Bacto yeast extract	1g
NaCl	1g
Agar Pouder	2g

Restriction Enzyme Digestion

Tatsui

	lysis2-1	XbaI	PstI	10xbuffer	BSA	MilliQ	total
2 cuts	10µL	1µL	1µL	3µL	3µL	12µL	30µL
1 cut	2µL	0.2µL	--	1µL	1µL	5.8µL	10µL
1 cut	2µL	--	0.2µL	1µL	1µL	5.8µL	10µL
NC	2µL	--	--	1µL	1µ	6µL	10µL

at 37°C, for 1h

Murakami and Okazaki

	8/19 lysis1-1	XbaI	PstI	10xbuffer	BSA	MilliQ	total
2 cuts	2µL	0.5µL	0.8µL	2µL	2µL	13µL	20µL
1 cut	0.5µL	0.2µL	--	1µL	1µL	7.3µL	10µL
1 cut	0.5µL	--	0.2µL	1µL	1µL	7.3µL	10µL
NC	0.5µL	--	--	1µL	1µL	7.5µL	10µL

at 37°C, for 1h

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	RBS	0.3	lysis2	7.4	4
NC	RBS	0.3	MilliQ	7.4	4

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	8/19 lysis1	XbaI	PstI
3	8/19 lysis1	XbaI	--
4	8/19 lysis1	--	PstI
5	8/19 lysis1	--	--
6	8/19 lysis1(Gel Extraction Product)	XbaI	PstI

Transformation

Murata and Kojima

Name	Sample	Competent Cells	Plate
8/18 Plux+RBS-GFP-DT	2µL	20µL	CP
8/18 Plux(NC)	2µL	20µL	CP
8/19 RBS+lysis2	2µL	20µL	Amp
8/15 Pbad-araC	2µL	20µL	Kan

Incubate at 37°C

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	8/19 lysis1 ①	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
8/19 lysis1 ① (XbaI & PstI)	87.8	1.06	0.82

Liquid Culture

Ashida

Sample	medium
8/18 Ptet -1	Plusgrow medium(+CP)
8/18 Ptet -2	Plusgrow medium(+CP)
8/18 RBS-tetR-DT -1	Plusgrow medium(+CP)
8/18 RBS-tetR-DT -2	Plusgrow medium(+CP)
8/18 J23100-GFP -1	Plusgrow medium(+Amp)
8/18 J23100-luxR -1	Plusgrow medium(+Amp)
8/18 J23100-luxR -2	Plusgrow medium(+Amp)
8/18 J23100-lacZα -1	Plusgrow medium(+Amp)
8/18 RBS-lysis3 -1	Plusgrow medium(+Amp)
8/18 RBS-lysis3 -2	Plusgrow medium(+Amp)

Master Plate

Ashida

Number	Use LB plate(+Amp)
1	8/18 J23100-GFP -1
2	8/18 J23100-luxR -1
3	8/18 J23100-luxR -2
4	8/18 J23100-lacZα -1
5	8/18 RBS-lysis3 -1
6	8/18 RBS-lysis3 -2

Number	Use LB plate(+CP)
1	8/18 Ptet -1
1	8/18 Ptet -2
3	8/18 RBS-tetR-DT -1
4	8/18 RBS-tetR-DT -2

Aug 20

Miniprep

Kojima

DNA	concentration[µg/µL]	260/280	260/230
8/18 Ptet -1	220	1.69	1.87
8/18 Ptet -2	210	1.69	1.71
8/18 RBS-tetR-DT -1	236	1.63	1.69
8/18 RBS-tetR-DT -2	182	1.65	1.98
8/18 J23100-RBS-GFP-DT -1	334	1.71	2.12
8/18 J23100-RBS-luxR-DT -1	440	1.67	1.60
8/18 J23100-RBS-luxR-DT -2	344	1.68	1.83
8/18 J23100-RBS-lacZα-DT -1	280	1.70	1.81
8/18 RBS-lysis3 -1	282	1.67	1.76
8/18 RBS-lysis3 -2	212	1.30	1.35

LB Medium Plate

Okazaki

volume	200mL
Bacto Trypton	2g
Bacto yeast extract	1g
Nacl	1g
Agar Powder	2g
Amp	40µl

1. Measuring reagents and put they in the Erlenmeyer flask.
2. diluting in measuring cylinder to 200ml total.
3. autoclaving.
4. adding ampicirine 40ml.

Ristriction Enzyme Digestion

Kojima and Ashida

	8/20 Ptet -1(220µg/mL)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2 cuts	4.5	1	1	3	3	17.5	30
1 cut	0.5	0.2	0	1	1	7.3	10
1 cut	0.5	0	0.2	1	1	7.3	10
NC	0.5	0	0	1	1	7.5	10

	8/20 RBS-tetR-DT -2(182µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
2 cuts	5.5	1	1	3	19.5	30
1 cut	0.6	0.2	0	1	8.2	10
1 cut	0.6	0	0.2	1	8.2	10
NC	0.6	0	0	1	8.4	10

	8/20 J23100-RBS-GFP-DT(334µg/ml)	EcoRI	SpeI	10x buffer H	MilliQ	total
2 cuts	3.0	1	1	3	22	30
1 cut	0.3	0.2	0	1	8.5	10
1 cut	0.3	0	0.2	1	8.5	10
NC	0.3	0	0	1	8.7	10

	8/20 J23100-RBS-luxR-DT -2(344µg/mL)	EcoRI	XbaI	BSA	10x buffer M	MilliQ	total
2 cuts	2.9	1	1	3	3	19.1	30
1 cut	0.3	0.2	0	1	1	7.5	10
1 cut	0.3	0	0.2	1	1	7.5	10
NC	0.3	0	0	1	1	7.7	10

	8/20 RBS-lysis3 -1(282µg/mL)	EcoRI	SpeI	10x buffer H	MilliQ	total
2 cuts	3.5	1	1	3	21.5	30
1 cut	0.4	0.2	0	1	8.4	10
1 cut	0.4	0	0.2	1	8.4	10
NC	0.4	0	0	1	8.6	10

	8/17 DT -1(188µg/mL)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2 cuts	5.3	1	1	3	3	16.7	30
1 cut	0.3	0.2	0	1	1	7.5	10
1 cut	0.3	0	0.2	1	1	7.5	10
NC	0.3	0	0	1	1	7.7	10

Transformation

Nakamoto

Name	Sample	Competent Cells(XL10-gold)	Total	Plate
tRNA-Spinach-tRNA	1µL	10µL	11µL	Amp
tetR-aptamer 12_PC(076)	1µL	10µL	11µL	Amp
tetR-aptamer 12_1R(113)	1µL	10µL	11µL	Amp
tetR-aptamer 12_1M(105)	1µL	10µL	11µL	Amp
pT181 attenuator	1µL	10µL	11µL	Amp
Fusin1 attenuator	1µL	10µL	11µL	Amp
Fusion3m2 attenuator	1µL	10µL	11µL	Amp
pT181 antisense	1µL	10µL	11µL	Amp
Fusion1 antisense	1µL	10µL	11µL	Amp
Fusion6 antisense	1µL	10µL	11µL	Amp

incubate 37°C overnight 20:34~

Electrophoresis

Kojima

Lane	Sample	Enzyme1	Enzyme2
1	8/20 Ptet -1	EcoRI	XbaI
2	8/20 Ptet -1	EcoRI	--
3	8/20 Ptet -1	--	XbaI
4	8/20 Ptet -1	--	--
5	8/20 RBS-tetR-DT -2	EcoRI	SpeI
6	8/20 RBS-tetR-DT -2	EcoRI	--
7	8/20 RBS-tetR-DT -2	--	SpeI
8	8/20 RBS-tetR-DT -2	--	--
9	1kbp ladder	--	--
10	8/20 J23100-RBS-GFP-DT -1	EcoRI	SpeI
11	8/20 J23100-RBS-GFP-DT -1	EcoRI	--
12	8/20 J23100-RBS-GFP-DT -1	--	SpeI
13	8/20 J23100-RBS-GFP-DT -1	--	--
14	8/20 J23100-RBS-luxR-DT -1	EcoRI	XbaI
15	8/20 J23100-RBS-luxR-DT -1	EcoRI	--
16	8/20 J23100-RBS-luxR-DT -1	--	XbaI
17	8/20 J23100-RBS-luxR-DT -1	--	--
18	1kbp ladder	--	--
19	8/20 RBS-lysis3 -1	EcoRI	SpeI
20	8/20 RBS-lysis3 -1	EcoRI	--
21	8/20 RBS-lysis3 -1	--	SpeI
22	8/20 RBS-lysis3 -1	--	--
23	1kbp ladder	--	--
24	8/17 DT	EcoRI	XbaI
25	8/17 DT	EcoRI	--
26	8/17 DT	--	XbaI
27	8/17 DT	--	--

Aug 21

Plusgrow Medium

Tatsui

material

8/17 Plusgrow medium 50ml
7/22 5000x Ampicillin 10µl

Measuring materials and putting them in a 50ml tube

Liquid culture

Tatsui

Sample	medium
8/20 tRNA-spinach -(1)	8/21 Plusgrow medium(+Amp)
8/20 tRNA-spinach -(2)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(1)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(2)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(1)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(2)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(1)	8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(2)	8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(1)	8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(2)	8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(1)	8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(2)	8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(1)	8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(2)	8/21 Plusgrow medium(+Amp)

incubate 37°C 10hour

Gel Extraction

Nakamoto and Tatsui

Lane	DNA	Enzyme
1	1kbp ladder	--
2	8/20 Ptet -(1)	EcoRI & XbaI
3	8/20 Ptet -(1)	EcoRI & XbaI
5	8/20 RBS-tetR-DT -(2)	EcoRI & SpeI
6	8/20 RBS-tetR-DT -(2)	EcoRI & SpeI
8	8/20 Pconst-RBS-GFP-DT -(1)	EcoRI & SpeI
9	8/20 Pconst-RBS-GFP-DT -(1)	EcoRI & SpeI
11	8/20 RBS-lysis3 -(1)	EcoRI & SpeI
12	8/20 RBS-lysis3 -(1)	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
Ptet (EcoRI & XbaI)	21	1.55	0.78
RBS-tetR-DT (EcoRI & SpeI)	12	1.45	0.62
Pconst-RBS-GFP-DT (EcoRI & SpeI)	11	1.36	0.60
RBS-lysis3 (EcoRI & SpeI)	10	1.26	0.50

Ristriction Enzyme Digestion

Kojima and Nakamoto

	8/20 Ptet-(1)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2 cuts	4.5	1	1	3	3	17.5	30
1 cut	0.5	0.2	0	1	1	7.3	10
1 cut	0.5	0	0.2	1	1	7.3	10
NC	0.5	0	0	1	1	7.5	10

	8/20 Pconst-RBS-luxR-DT-(2)	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2 cuts	2.9	1	1	3	3	19.1	30
1 cut	0.3	0.2	0	1	1	7.5	10
1 cut	0.3	0	0.2	1	1	7.5	10
NC	0.3	0	0	1	1	7.7	10

	8/17 DT	EcoRI	XbaI	10x BSA	10x buffer M	MilliQ	total
2 cuts	5.3	1	1	3	3	16.7	30
1 cut	0.5	0.2	0	1	1	7.3	10
1 cut	0.5	0	0.2	1	1	7.3	10
NC	0.5	0	0	1	1	7.5	10

incubate at 37°C for 1h

Electrophoresis

Kojima and Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	8/20 Ptet -(1)	EcoRI	XbaI
3	8/20 Ptet -(1)	EcoRI	--
4	8/20 Ptet -(1)	--	XbaI
5	8/20 Ptet -(1)	--	--
6	8/20 J23100-RBS-luxR-DT -(2)	EcoRI	XbaI
7	8/20 J23100-RBS-luxR-DT -(2)	EcoRI	--
8	8/20 J23100-RBS-luxR-DT -(2)	--	XbaI
9	8/20 J23100-RBS-luxR-DT -(2)	--	--
10	8/20 DT	EcoRI	XbaI
11	8/20 DT	EcoRI	--
12	8/20 DT	--	XbaI
13	8/20 DT	--	--
14	1kbp ladder	--	--
15	8/20 Ptet -(1)	EcoRI	XbaI

The reason why lane 2 and 15 were same is that the well of lane 2 might be broken.

Kojima

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	8/20 J23100-RBS-luxR-DT -(2)	EcoRI	XbaI
3	8/20 DT	EcoRI	XbaI

Gel Extraction

Honda

Lane	DNA	Enzyme
1	1kb ladder	--
2	8/21 J23100-RBS-luxR-DT 25µL	EcoRI+XbaI
3	8/21 J23100-RBS-luxR-DT 25µL	EcoRI+XbaI
4	--	--
5	8/21 Ptet 25µL	EcoRI+XbaI
6	8/21 Ptet 25µL	EcoRI+XbaI

Electrophoresis

Honda

Lane	Sample	EcoRI	XbaI
1	1kb ladder	--	--
2	8/21 DT	+	+
3	8/21 DT	+	--
4	8/21 DT	--	+
5	8/21 DT	--	--

Ligation

No name

state	Vector		Inserter		Ligation High ver.2	total
experiment	8/21 Pcn-RBS-luxR-DT(+Amp)	2.3	8/21 Pcn-RBS-GFP-DT	1.7	2	6
NC	8/21 Pcn-RBS-luxR-DT(+Amp)	2.3	MilliQ	1.7	2	6
experiment	8/18 Plux(+CP)	1.1	8/19 RBS-GFP-DT	5	3.1	9.2
NC	8/18 Plux(+CP)	1.1	MilliQ	5	3.1	9.2
experiment	8/18 RBS(+Amp)	2.2	8/19 lysis1	6.0	4.1	12.3
experiment	8/18 RBS(+Amp)	2.2	8/19 lysis2	3.6	2.9	8.7
NC	8/18 RBS(+Amp)	2.2	MilliQ	3.6	2.9	8.7
experiment	8/21 Ptet	1.5	8/20 RBS-tetR-DT (2)	2.1	1.8	5.4
NC	8/21 Ptet	1.5	MilliQ	2.1	1.8	5.4

Transformation

Okazaki

Name	Sample	Competent Cells	Total	Plate
8/21 Pcon-RBS-GFP-DT+Pcon-RBS-GFP-DT	1µL	10µL	11µL	Amp
8/21 Pcon-RBS-GFP-DT NC	1µL	10µL	11µL	Amp
8/18 RBS+8/19 lysis1	1µL	10µL	11µL	Amp
8/18 RBS+8/19 lysis2	1µL	10µL	11µL	Amp
8/18 RBS NC	1µL	10µL	11µL	Amp
8/18 Plux+8/18 RBS-GFP-DT	1µL	10µL	11µL	CP
8/18 Plux+8/18 RBS-GFP-DT	1µL	10µL	11µL	CP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2)	1µL	10µL	11µL	CP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2) NC	1µL	10µL	11µL	CP
8/21 pSB1C3	1µL	10µL	11µL	CP

LB Medium Plate

Gel Extraction

Okazaki

Lane	DNA	Enzyme
1	1kb ladder	--
2	--	--
3	8/21 DT	EcoRI & XbaI
4	8/21 DT	EcoRI & XbaI

Miniprep

Tatsui, Kojima, and Nakamoto

DNA	concentration [µg/mL]	260/280	260/230
tRNA-spinach-(1)	234	1.64	2.14
tRNA-spinach-(2)	440	1.60	1.91
tetR-aptamer 12-p(1076)-(1)	150	1.61	2.03
tetR-aptamer 12-p(1076)-(2)	374	1.45	1.66
tetR-aptamer 12-1R(113)-(1)	304	1.68	2.21
tetR-aptamer 12-1R(113)-(2)	382	1.47	1.66
tetR-aptamer 12-1M(105)-(1)	374	1.68	2.20
tetR-aptamer 12-1M(105)-(2)	286	1.31	1.43
PT181 attenuator-(1)	330	1.66	2.11
PT181 attenuator-(2)	270	1.68	2.18
Fusion1 attenuator-(1)	306	1.67	2.20
Fusion1 attenuator-(2)	282	1.60	1.94
Fusion3m2 attenuator-(1)	332	1.68	2.25
Fusion3m2 attenuator-(2)	326	1.67	2.10
PT181 antisense-(1)	178	1.64	1.68
PT181 antisense-(2)	116	1.63	1.95
Fusion1 antisense-(1)	300	1.67	2.21
Fusion1 antisense-(2)	164	1.42	1.46
Fusion6 antisense-(1)	336	1.65	2.18
Fusion6 antisense-(2)	192	1.65	2.01

Aug 22

Liquid culture

Nakamoto

Sample	Medium
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (1)	8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (2)	8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (1)	8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (2)	8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (1)	8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (2)	8/21 Plusgrow medium (+Amp)
8/21 RBS control (1)	8/21 Plusgrow medium (+Amp)
8/21 RBS control (2)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (1)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (2)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (1)	8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (2)	8/21 Plusgrow medium (+Amp)
8/21 Plux-RBS-GFP-DT (1)	8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (1)	8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (2)	8/21 Plusgrow medium (+Amp)

incubate 37°C 10hour

Colony PCR

Kojima

Sample	base pair
8/21 RBS-lysis1(1)	400
8/21 RBS-lysis1(2)	400
8/21 RBS control(1)	--
8/21 RBS control(2)	--
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

Sample	base pair
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (1)	2143
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (2)	2143
8/21 Pcon-RBS-luxR control(1)	--
8/21 Pcon-RBS-luxR control(2)	--
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycles

Sample	base pair
8/21 Ptet-RBS-tetR-DT (1)	1216
8/21 Ptet-RBS-tetR-DT (2)	1216
8/21 Ptet control (1)	--
8/21 Ptet control (2)	--
8/21 Plux-RBS-GFP-DT (1)	1227
8/21 pSB1C3(BBa_J04450) (1)	1353
8/21 pSB1C3(BBa_J04450) (2)	1353
negative control	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Restriction Enzyme Digestion

Tatsui, Kojima, Honda and Ashida

	Fusion1 antisense (1) (300 µg/mL)	EcoRI	SpeI	buffer H	MilliQ	total
2 cuts	3.3	1	1	3	21.7	30
1 cut	0.7	0.2	0	1	8.1	10
1 cut	0.7	0	0.2	1	8.1	10
NC	0.7	0	0	1	8.3	10

	Fusion1 antisense (1) (300 µg/mL)	XbaI	PstI	buffer M	BSA	MilliQ	total
2 cuts	3.3	1	1	3	3	18.7	30
1 cut	0.7	0.2	0	1	1	7.1	10
1 cut	0.7	0	0.2	1	1	7.1	10
NC	0.7	0	0	1	1	7.3	10

	Fusion6 antisense (1) (336 µg/mL)	EcoRI	SpeI	buffer H	MilliQ	total
2 cuts	3	1	1	3	22	30
1 cut	0.6	0.2	0	1	8.2	10
1 cut	0.6	0	0.2	1	8.2	10
NC	0.6	0	0	1	8.4	10

	Fusion6 antisense (1) (336 µg/mL)	XbaI	PstI	buffer M	BSA	MilliQ	total
2 cuts	3	1	1	3	3	19	30
1 cut	0.6	0.2	0	1	1	7.2	10
1 cut	0.6	0	0.2	1	1	7.2	10
NC	0.6	0	0	1	1	7.4	10

	pT181 antisense (1) (178 µg/mL)	EcoRI	SpeI	buffer H	MilliQ	total
2 cuts	5.6	1	1	3	19.4	30
1 cut	1.1	0.2	0	1	7.7	10
1 cut	1.1	0	0.2	1	7.7	10
NC	1.1	0	0	1	7.9	10

	pT181 antisense (1) (178 µg/mL)	XbaI	PstI	buffer M	BSA	MilliQ	total
2 cuts	5.6	1	1	3	3	16.4	30
1 cut	1.1	0.2	0	1	1	6.7	10
1 cut	1.1	0	0.2	1	1	6.7	10
NC	1.1	0	0	1	1	6.9	10

	pT181 attenuator (1)	EcoRI	SpeI	buffer H	MilliQ	total
2 cuts	3	1	1	3	22	30
1 cut	0.6	0.2	0	1	8.2	10
1 cut	0.6	0	0.2	1	8.2	10
NC	0.6	0	0	1	8.4	10

	pT181 attenuator (1)	XbaI	PstI	buffer M	BSA	MilliQ	total
2 cuts	3	1	1	3	3	19	30
1 cut	0.6	0.2	0	1	1	7.2	10
1 cut	0.6	0	0.2	1	1	7.2	10
NC	0.6	0	0	1	1	7.4	10

	Fusion1 attenuator (1)	EcoRI	SpeI	buffer H	MilliQ	total
2 cuts	3.3	1	1	3	21.7	30
1 cut	0.7	0.2	0	1	8.1	10
1 cut	0.7	0	0.2	1	8.1	10
NC	0.7	0	0	1	8.3	10

	Fusion1 attenuator (1)	XbaI	PstI	buffer M	BSA	MilliQ	total
2 cuts	3.3	1	1	3	3	18.7	30
1 cut	0.7	0.2	0	1	1	7.1	10
1 cut	0.7	0	0.2	1	1	7.1	10
NC	0.7	0	0	1	1	7.3	10

	Fusion3m2 attenuator (1)	EcoRI	SpeI	buffer H	MilliQ	total
2 cuts	3	1	1	3	22	30
1 cut	0.6	0.2	0	1	8.2	10
1 cut	0.6	0	0.2	1	8.2	10
NC	0.6	0	0	1	8.4	10

	Fusion3m2 attenuator (1)	XbaI	PstI	buffer M	BSA	MilliQ	total
2 cuts	3	1	1	3	3	19	30
1 cut	0.6	0.2	0	1	1	7.2	10
1 cut	0.6	0	0.2	1	1	7.2	10
NC	0.6	0	0	1	1	7.4	10

	Spinach (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2 cuts	2.3	0.5	0.5	3	0.3	23.4	30
1 cut	0.5	0.1	0	1	0.1	8.3	10
1 cut	0.5	0	0.1	1	0.1	8.3	10
NC	0.5	0	0	1	0.1	8.4	10

	tetR aptamer 12_P (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2 cuts	2.7	0.5	0.5	3	0.3	23	30
1 cut	0.5	0.1	0	1	0.1	8.3	10
1 cut	0.5	0	0.1	1	0.1	8.3	10
NC	0.5	0	0	1	0.1	8.4	10

	tetR aptamer 12_1R (2)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2 cuts	2.6	0.5	0.5	3	0.3	23.1	30
1 cut	0.5	0.1	0	1	0.1	8.3	10
1 cut	0.5	0	0.1	1	0.1	8.3	10
NC	0.5	0	0	1	0.1	8.4	10

	tetR aptamer 12_1M (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2 cuts	2.7	0.5	0.5	3	0.3	23	30
1 cut	0.5	0.1	0	1	0.1	8.3	10
1 cut	0.5	0	0.1	1	0.1	8.3	10
NC	0.5	0	0	1	0.1	8.4	10

	J23100	SpeI	PstI	buffer B	BSA	MilliQ	total
2 cuts	7	0.5	0.5	3	0.3	18.7	30
1 cut	1.4	0.1	0	1	0.1	7.4	10
1 cut	1.4	0	0.1	1	0.1	7.4	10
NC	1.4	0	0	1	0.1	7.5	10

	Plac (2)	SpeI	PstI	buffer B	BSA	MilliQ	total
2 cuts	11.2	0.5	0.5	3	0.3	14.5	30
1 cut	2.2	0.1	0	1	0.1	6.6	10
1 cut	2.2	0	0.1	1	0.1	6.6	10
NC	2.2	0	0	1	0.1	6.7	10

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	RBS-lysis1 -(1)
3	RBS-lysis1 -(2)
4	RBS-lysis2 -(1)
5	RBS-lysis2 -(2)
6	RBS(NC) -(1)
7	RBS(NC) -(2)
8	NC

Lane	Sample
1	1kb ladder
2	Ptet-RBS-tetR-DT -(1)
3	Ptet-RBS-tetR-DT -(2)
4	Ptet(NC) -(1)
5	Ptet(NC) -(2)
6	Plux-RBS-GFP-DT -(1)
7	pSB1C3 -(1)
8	pSB1C3 -(2)
9	NC

Lane	Sample
1	1kb ladder
2	Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(1)
3	Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(2)
4	Pcon-RBS-luxR -(1)
5	Pcon-RBS-luxR -(2)
6	NC
7	1kb ladder

Miniprep

Ashida

DNA	concentration[µg/mL]	260/280	260/230
8/22 RBS-lysis (1)	128	1.77	2.29
8/22 RBS-lysis (2)	96	1.80	2.07
8/22 pSB1C3 (1)	108	1.55	1.45
8/22 pSB1C3 (2)	130	1.69	1.76

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	8/22 pT181 attenuator (1)	EcoRI	SpeI
3	8/22 pT181 attenuator (1)	EcoRI	--
4	8/22 pT181 attenuator (1)	--	SpeI
5	8/22 pT181 attenuator (1)	--	--
6	8/22 pT181 attenuator (1)	XbaI	PstI
7	8/22 pT181 attenuator (1)	XbaI	--
8	8/22 pT181 attenuator (1)	--	PstI
9	8/22 pT181 attenuator (1)	--	--
10	8/22 Fusion6 antisense (1)	EcoRI	SpeI
11	8/22 Fusion6 antisense (1)	EcoRI	--
12	8/22 Fusion6 antisense (1)	--	SpeI
13	8/22 Fusion6 antisense (1)	--	--
14	100bp	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	8/22 Fusion6 antisense (1)	XbaI	PstI
3	8/22 Fusion6 antisense (1)	XbaI	--
4	8/22 Fusion6 antisense (1)	--	PstI
5	8/22 Fusion6 antisense (1)	--	--
6	8/22 Fusion1 antisense (1)	EcoRI	SpeI
7	8/22 Fusion1 antisense (1)	EcoRI	--
8	8/22 Fusion1 antisense (1)	--	SpeI
9	8/22 Fusion1 antisense (1)	--	--
10	8/22 Fusion1 antisense (1)	XbaI	PstI
11	8/22 Fusion1 antisense (1)	XbaI	--
12	8/22 Fusion1 antisense (1)	--	PstI
13	8/22 Fusion1 antisense (1)	--	--
14	100bp	--	--

Aug 23

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	pT181 antisense	EcoRI	SpeI
2	pT181 antisense	EcoRI	--
3	pT181 antisense	--	SpeI
4	pT181 antisense	--	--
5	pT181 antisense	XbaI	PstI
6	pT181 antisense	XbaI	--
7	pT181 antisense	--	PstI
8	pT181 antisense	--	--
9	100bp ladder	--	--
10	Fusion1 attenuator	EcoRI	SpeI
11	Fusion1 attenuator	EcoRI	--
12	Fusion1 attenuator	--	SpeI
13	Fusion1 attenuator	--	--
14	Fusion1 attenuator	XbaI	PstI
15	Fusion1 attenuator	XbaI	--
16	Fusion1 attenuator	--	PstI
17	Fusion1 attenuator	--	--

Lane	Sample	Enzyme1	Enzyme2
1	Fusion3m2 attenuator	EcoRI	SpeI
2	Fusion3m2 attenuator	EcoRI	--
3	Fusion3m2 attenuator	--	SpeI
4	Fusion3m2 attenuator	--	--
5	Fusion3m2 attenuator	XbaI	PstI
6	Fusion3m2 attenuator	XbaI	--
7	Fusion3m2 attenuator	--	PstI
8	Fusion3m2 attenuator	--	--
9	100bp ladder	--	--
10	tetR aptamer 12_1M	EcoRI	SpeI
11	tetR aptamer 12_1M	EcoRI	--
12	tetR aptamer 12_1M	--	SpeI
13	tetR aptamer 12_1M	--	--
14	tetR aptamer 12_P	EcoRI	SpeI
15	tetR aptamer 12_P	EcoRI	--
16	tetR aptamer 12_P	--	SpeI
17	tetR aptamer 12_P	--	--

Kojima and Honda

Lane	Sample	Enzyme1	Enzyme2
1	Plac	SpeI	PstI
2	Plac	SpeI	--
3	Plac	--	PstI
4	Plac	--	--
5	Spinach	EcoRI	SpeI
6	Spinach	EcoRI	--
7	Spinach	--	SpeI
8	Spinach	--	--
9	100bp ladder	--	--
10	J23100	SpeI	PstI
11	J23100	SpeI	--
12	J23100	--	PstI
13	J23100	--	--
14	tetR aptamer 12_1R	EcoRI	SpeI
15	tetR aptamer 12_1R	EcoRI	--
16	tetR aptamer 12_1R	--	SpeI
17	tetR aptamer 12_1R	--	--

Restriction Enzyme Digestion

Nakamoto and Kojima

	Fusion6 antisense (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2 cuts	3	0.5	0.5	3	0.3	22.7	30
1 cut	0.6	0.1	0	1	0.1	8.2	10
1 cut	0.6	0	0.1	1	0.1	8.2	10
NC	0.6	0	0	1	0.1	8.3	10

	Fusion6 antisense (1)	XbaI	PstI	buffer D	BSA	MilliQ	total
2 cuts	3	0.5	0.5	3	0.3	22.7	30
1 cut	0.6	0.1	0	1	0.1	8.2	10
1 cut	0.6	0	0.1	1	0.1	8.2	10
NC	0.6	0	0	1	0.1	8.3	10

	Fusion1 antisense (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2 cuts	3.3	0.5	0.5	3	0.3	22.4	30
1 cut	0.7	0.1	0	1	0.1	8.1	10
1 cut	0.7	0	0.1	1	0.1	8.1	10
NC	0.7	0	0	1	0.1	8.2	10

	pT181 antisense (1)	EcoRI	SpeI	buffer B	BSA	MilliQ	total
2 cuts	5.6	0.5	0.5	3	0.3	20.1	30
1 cut	1.1	0.1	0	1	0.1	7.7	10
1 cut	1.1	0	0.1	1	0.1	7.7	10
NC	1.1	0	0	1	0.1	7.8	10

	pT181 antisense (1)	XbaI	PstI	buffer D	BSA	MilliQ	total
2 cuts	5.6	0.5	0.5	3	0.3	20.1	30
1 cut	1.1	0.1	0	1	0.1	7.7	10
1 cut	1.1	0	0.1	1	0.1	7.7	10
NC	1.1	0	0	1	0.1	7.8	10

incubate at 37°C from 17:29.

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	pT181 attenuator	EcoRI	SpeI
3	pT181 attenuator	XbaI	PstI
4	Fusion3m2 attenuator	EcoRI	SpeI
5	Fusion1 attenuator	XbaI	PstI
6	Fusion1 attenuator	EcoRI	SpeI
7	Fusion3m2 attenuator	XbaI	PstI
8	pT181 antisense	EcoRI	SpeI
9	pT181 antisense	XbaI	PstI
10	Fusion1 antisense	EcoRI	SpeI
11	Fusion1 antisense	XbaI	PstI
12	Fusion6 antisense	EcoRI	SpeI
13	Fusion6 antisense	XbaI	PstI
14	tetR aptamer 12_1M	EcoRI	SpeI
15	tetR aptamer 12_P	EcoRI	SpeI
16	tetR aptamer 12_1R	EcoRI	SpeI
17	100bp	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	Spinach	EcoRI	SpeI
3	J23100	SpeI	PstI
4	Plac	SpeI	PstI
5	100bp	--	--
6	J23100	SpeI	PstI

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	Fusion6 antisense	EcoRI	SpeI
3	Fusion6 antisense	EcoRI	--
4	Fusion6 antisense	--	SpeI
5	Fusion6 antisense	--	--
6	pT181 antisense	XbaI	PstI
8	100bp	--	--
9	100bp	--	--
10	Fusion6 antisense	XbaI	PstI
11	Fusion6 antisense	XbaI	--
12	Fusion6 antisense	--	PstI
13	Fusion6 antisense	--	--
14	pT181 antisense	XbaI	--
16	100bp	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp	--	--
2	Fusion1 antisense	EcoRI	SpeI
3	Fusion1 antisense	EcoRI	--
4	Fusion1 antisense	--	SpeI
5	Fusion1 antisense	--	--
6	pT181 antisense	--	PstI
8	100bp	--	--
9	100bp	--	--
10	pT181 antisense	EcoRI	SpeI
11	pT181 antisense	EcoRI	--
12	pT181 antisense	--	SpeI
13	pT181 antisense	--	--
14	pT181 antisense	--	--
16	100bp	--	--

Gel Extraction

Nakamoto, Ashida, Kojima, Okazaki & Matsumoto

Lane	DNA	Enzyme
1	100bp ladder	--
2	pT181 attenuator	XbaI & PstI
3	pT181 attenuator	XbaI & PstI
5	Fusion3m2 attenuator	EcoRI & SpeI
6	Fusion3m2 attenuator	EcoRI & SpeI
8	Fusion1 attenuator	XbaI & PstI
9	Fusion1 attenuator	XbaI & PstI
11	Fusion1 attenuator	EcoRI & SpeI
12	Fusion1 attenuator	EcoRI & SpeI

Lane	DNA	Enzyme
1	100bp ladder	--
2	Fusion3m2 attenuator	XbaI & PstI
3	Fusion3m2 attenuator	XbaI & PstI
5	pT181 antisense	EcoRI & SpeI
6	pT181 antisense	EcoRI & SpeI
8	pT181 antisense	XbaI & PstI
9	pT181 antisense	XbaI & PstI
11	Fusion1 antisense	XbaI & PstI
12	Fusion1 antisense	XbaI & PstI

Lane	DNA	Enzyme
1	100bp ladder	--
2	Fusion6 attenuator	XbaI & PstI
3	Fusion6 attenuator	XbaI & PstI
5	aptamer 12_1M	EcoRI & SpeI
6	aptamer 12_1M	EcoRI & SpeI

Lane	DNA	Enzyme
1	100bp ladder	--
2	aptamer 12_P	EcoRI & SpeI
3	aptamer 12_P	EcoRI & SpeI
5	aptamer 12_1R	EcoRI & SpeI
6	aptamer 12_1R	EcoRI & SpeI

Ashida

Lane	DNA	Enzyme
1	100bp ladder	--
2	Fusion6 antisense	EcoRI & SpeI
3	Fusion6 antisense	EcoRI & SpeI
5	Fusion6 attenuator	XbaI & PstI
6	Fusion6 attenuator	XbaI & PstI
8	Fusion1 antisense	EcoRI & SpeI
9	Fusion1 antisense	EcoRI & SpeI
11	pT181 antisense	EcoRI & SpeI
12	pT181 antisense	EcoRI & SpeI

Lane	DNA	Enzyme
1	100bp ladder	--
3	pT181 antisense	XbaI & PstI
4	pT181 antisense	XbaI & PstI

Lane	DNA	Enzyme
1	100bp ladder	--
3	Plac	SpeI & PstI
4	Plac	SpeI & PstI

Lane	DNA	Enzyme
1	100bp ladder	--
3	J23100	SpeI & PstI
5	Spinach	EcoRI & SpeI
6	Spinach	EcoRI & SpeI

Aug 24

Gel Extraction

Ashida

Name	concentration[µg/mL]	260/280	260/230
8/22 aptamer 12_P (EcoRI & SpeI)	88	21.3	--
8/22 aptamer 12_1R (EcoRI & SpeI)	63	23.1	--
8/22 aptamer 12_1M (EcoRI & SpeI)	40	22.0	--
8/23 Fusion6 antisense (EcoRI & SpeI)	6.0	1.60	0.17
8/23 Fusion6 antisense (XbaI & PstI)	6.0	1.71	0.25
8/23 pT181 antisense (EcoRI & SpeI)	6.0	2.22	0.15
8/23 pT181 antisense (XbaI & PstI)	6.0	2.10	0.04
8/22 Plac (SpeI & PstI)	6.0	1.47	0.09
8/22 Fusion1 antisense (EcoRI & SpeI)	10	2.16	0.01
8/22 J23100 (SpeI & PstI)	6.0	1.52	0.35

Name	concentration[µg/mL]	260/280	260/230
8/22 Spinach (EcoRI & SpeI)	8.0	1.15	0.77
8/22 Fusion6 antisense (XbaI & PstI)	44	1.07	0.96
8/22 Fusion1 antisense (XbaI & PstI)	2	--	1.00
8/22 pT181 antisense (XbaI & PstI)	3.	2.01	0.40
8/22 pT181 antisense (EcoRI & SpeI)	8	1.13	0.82
8/22 Fusion3m2 attenuator (XbaI & PstI)	5	2.27	1.50
8/22 Fusion3m2 attenuator (EcoRI & SpeI)	34	1.16	1.02
8/22 Fusion1 attenuator (XbaI & PstI)	14	1.40	0.62
8/22 Fusion1 attenuator (EcoRI & SpeI)	16	1.27	0.61
8/22 pT181 attenuator (XbaI & PstI)	5	--	0.88

These samples were performed ethanol ethanl precipitation.

Restriction Enzyme Digestion

No name

	tetR aptamer 12_P(2)	EcoRI	SpeI	10x Buffer B	100x BSA	MilliQ	total
2 cuts	2.7µL	0.5µL	0.5µL	3µL	0.3µL	23µL	30µL
NC	0.3µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

	tetR aptamer 12_1R(2)	EcoRI	SpeI	10x Buffer B	100x BSA	MilliQ	total
2 cuts	2.6µL	0.5µL	0.5µL	3µL	0.3µL	23.1µL	30µL
NC	0.3µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

	tetR aptamer 12_M(1)	EcoRI	SpeI	10x Buffer B	100x BSA	MilliQ	total
2 cuts	2.7µL	0.5µL	0.5µL	3µL	0.3µL	23µL	30µL
NC	0.3µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

Master Plate

No name

Number	Use LB plate(+Kan)
1	entA- #1
2	entA- #1
3	entA- #1
4	entA- #2
5	entA- #2
6	entA- #2

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	-	-
2	tetR aptamer 12_P	EcoRI	SpeI
3	tetR aptamer 12_P	-	-
4	tetR aptamer 12_1R	EcoRI	SpeI
5	tetR aptamer 12_1R	-	-
6	tetR aptamer 12_1M	EcoRI	SpeI
7	tetR aptamer 12_1M	-	-
8	100bp ladder	-	-

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	-	-
2	tetR aptamer 12_P	EcoRI	SpeI
3	tetR aptamer 12_1R	EcoRI	SpeI
4	tetR aptamer 12_1M	EcoRI	SpeI
5	100bp ladder	-	-

Aug 26

Gel Extraction

Matsumoto and Ashida

Lane	DNA	Enzyme
1	100bp ladder	-
2	8/24 tetR aptamer 12_P	EcoRI & SpeI
4	8/24 tetR aptamer 12_1R	EcoRI & SpeI
6	8/24 tetR aptamer 12_M	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
tetR aptamer 12_P (EcoRI & SpeI)	2.3	0.38	0.13
tetR aptamer 12_1R (EcoRI & SpeI)	12.0	0.63	1.19
tetR aptamer 12_M (EcoRI & SpeI)	2.0	6.43	0.03

Ligation

Ashida

state	Vector		Inserter		Ligation High ver.2
experiment	8/21 DT	5.0	8/26 tetR aptamer 12_1R	2.0	3.5
experiment	8/21 DT	5.0	8/22 pT181 atteniator	5.0	5.0
experiment	8/21 DT	5.0	8/22 pT181 antisense	3.0	4.0
experiment	8/21 DT	5.0	8/22 Spinach	3.0	4.0
experiment	8/22 Plac	4.0	8/22 pT181 attenuator	5.0	4.5
experiment	8/22 J23100	4.0	8/22 pT181 attenuator	5.0	4.5
experiment	8/22 Plac	4.0	8/22 pT181 antisense	8.0	6.0
experiment	8/22 J23100	4.0	8/22 pT181 antisense	8.0	6.0

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
8/26 tetR aptamer 12_1R-DT	1µL	10µL	11µL	CP
8/26 pT181 attenuator-DT	1µL	10µL	11µL	CP
8/26 pT181 antisense-DT	1µL	10µL	11µL	CP
8/26 Spinach-DT	1µL	10µL	11µL	CP
8/26 Plac-pT181 attenuator	1µL	10µL	11µL	CP
8/26 Pcon-pT181 attenuator	1µL	10µL	11µL	Amp
8/26 Plac-pT181 antisense	1µL	10µL	11µL	CP
8/26 Pcon-pT181 antisense	1µL	10µL	11µL	Amp
Pλ-RBS-luxI-DT	1µL	10µL	11µ	Amp

Aug 27

Restriction Enzyme Digestion

No name

	8/21 pT181 attenuator-1(330µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	0.6µL	0µL	0µL	1µL	0.1µL	8.3µL	10µL

	8/17 DT-1(188µg/mL)	EcoRI	XbaI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	1.0µl	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/20 Pcon-RBS-luxR-DT-2(344µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	2.9µL	0.5µL	0.5µL	3.0µL	0.3µL	22.8µL	30µL
NC	0.6µl	0µL	0µL	1µL	0.1µL	8.3µL	10µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	8/21 pT181 attenuator	EcoI	SpeI
3	8/21 pT181 atteniator	--	--
4	8/17 DT-1	EcoRI	XbaI
5	8/17 DT-1	--	--
6	8/20 Pcon-RBS-luxR-DT-2	EcoI	SpeI
7	8/20 Pcon-RBS-luxR-DT-2	--	--
8	100bp ladder	--	--

Colony PCR

Kojima and Yoshida

Sample	base pair
8/26 Pλ-luxI(1)
8/26 Pλ-luxI(2)
NC

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	3min	30cycles

Lane	Sample
1	100bp ladder
2	Pλ-luxI-(1)
3	Pλ-luxI-(2)
4	NC

Liquid Culture

No name

Sample	medium
8/26 Pλ-RBS-luxI-DT-1	Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2	Plusgrow medium (+Amp)

Restriction Enzyme Digestion

No name

	8/21 pT181 attenuator-1(330µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	0.6µL	0µL	0µL	1µL	0.1µL	8.3µL	10µL

	8/17 DT-1(188µg/mL)	EcoRI	XbaI	10x BufferB	100x BSA	MilliQ	total
2 cuts	3.0µL	0.5µL	0.5µL	3.0µL	0.3µL	22.7µL	30µL
NC	1.0µl	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/20 Pcon-RBS-luxR-DT-2(344µg/mL)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	2.9µL	0.5µL	0.5µL	3.0µL	0.3µL	22.8µL	30µL
NC	0.6µl	0µL	0µL	1µL	0.1µL	8.3µL	10µL

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
	100bp ladder	--	--
1	pT181 attenuator	EcoRI	SpeI
2	pT181 attenuator	--	--
3	DT-(1)	EcoRI	XbaI
4	DT-(1)	--	--
5	Pcon-RBS-luxR-DT-(2)	EcoRI	SpeI
6	Pcon-RBS-luxR-DT-(2)	--	--
	100bp ladder	--	--

Gel Extraction

Inoue

Lane	DNA	Enzyme
1	100bpladder	--
2	pT181 attenuator-1	EcoRI&SpeI
3	pT181 attenuator-1	EcoRI&SpeI
4	DT-1	EcoRI&XbaI
5	DT-1	EcoRI&XbaI
6	Pcon-RBS-luxR-DT-2	EcoRI&SpeI
7	Pcon-RBS-luxR-DT-2	EcoRI&SpeI
8	100bpladder	--

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator-1(EcoRI&SpeI)	4.2	1.62	0.29
DT-1(EcoRI&XbaI)	17	1.88	0.88

Transformation

Kojima and Hirano

Name	Sample	Competent Cells	Total	Plate
8/26 tetR aptamer 12_1R-DT	1µL	10µL	11µL	CP
8/26 pT181 attenuator-DT	1µL	10µL	11µL	CP
8/26 pT181 antisense-DT	1µL	10µL	11µL	CP
8/26 Spinach-DT	1µL	10µL	11µL	CP
8/26 Plac-pT181 attenuator	1µL	10µL	11µL	CP
8/26 Pcon-pT181 attenuator	1µL	10µL	11µL	Amp
8/26 Plac-pT181 antisense	1µL	10µL	11µL	CP
8/26 Pcon-pT181 antisense	1µL	10µL	11µL	Amp

Liquid culture

No name

Sample	medium
8/16 Plux	Plusgrow medium(+CP)

Genome PCR

Yoshdia and Nakagawa

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	RpaA_fwd primer	RpaA_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	RpaB_fwd primer	RpaB_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	PkaiBC_fwd primer	PkaiBC_rev primer	MilliQ	total
0.5	0.5	2.5	2.5	1.5	0.75	0.75	16	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	50°C	68°C	--
2 min	10 sec	30 sec	38 sec	30 cycles

Ligation

No name

state	Vector		Inserter
experiment	8/27 DT (EcoRI & XbaI)	2.8	8/21 RBS-lysis3 (EcoRI & SpeI)
experiment	8/27 DT (EcoRI & XbaI)	2.8	8/27 pT181 attenuator (EcoRI & SpeI)

Samples were evaporeted used evaporator into about 3 µL.

sample	MilliQ	Ligation High	total
3	4	3.5	10.5

incubate overnight at 4 °C

Aug 28

Electrophoresis

No name

Lane	Sample
1	100bp
2	SasA
3	RpaA
4	RpaB
5	PkaiBC
6	PCR NC
7	100bp

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
8/27 Plux	173.8	1.95	1.45

Ligation

No name

state	Vector		Inserter
experiment	8/17 DT-1	3.0	8.0

Samples were evaporeted used evaporator into about 3 µL.

sample	MilliQ	Ligation High	total
3	4	3.5	10.5

incubate one hour at 16 °C

Gel Extraction

No name

Lane	DNA
1	100bpladder
3	SasA(8/27 Genome PCR production)
5	RpaA(8/27 Genome PCR production)
7	RpaB(8/27 Genome PCR production)
9	PkaiBC(8/27 Genome PCR production)

Name	concentration[µg/mL]	260/280	260/230
RpaA	15.4	1.88	0.03
SasA	21.6	1.66	0.79
PkaiBC	16.3	1.79	0.68
RpaB	26.7	1.77	0.82

Colony PCR

No name

Sample	base pair
8/21 RBS-lysis2-(3)	772
8/21 RBS-lysis2-(4)	772
8/21 RBS-lysis2-(5)	772
8/21 RBS-lysis2-(6)	772

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	48s	30cycles

Restriction Enzyme Digestion

No name

	8/22 RBS-lysis1-(1)	EcoRI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	9µL	0.5µL	0.5µL	3µL	0.3µL	16.7µL	30µL
NC	1µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/17 RBS-GFP-DT-(2)	XbaI	PstI	BufferD	BSA	MilliQ	total
2 cuts	4µL	0.5µL	0.5µL	3µL	0.3µL	21.7µL	30µL
NC	0.5µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	8/20 Pcon-RBS-GFP-DT-(1)	EcoRI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	3µL	0.5µL	0.5µL	3µL	0.3µL	22.7µL	30µL
NC	0.3µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

	8/20 Pcon-RBS-luxR-DT-(1)	EcoRI	XbaI	BufferD	BSA	MilliQ	total
2 cuts	2µL	0.5µL	0.5µL	3µL	0.3µL	23.7µL	30µL
NC	0.3µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

No name

	8/28 Plux	SpeI	PstI	BufferD	BSA	MilliQ	total
2 cuts	6µL	0.5µL	0.5µL	3µL	0.3µL	19.7µL	30µL
NC	1µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/20 RBS-tetR-DT-(1)	XbaI	PstI	BufferD	BSA	MilliQ	total
2 cuts	5µL	0.5µL	0.5µL	3µL	0.3µL	20.7µL	30µL
NC	0.5µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

incubated 37°C 1hour

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	8/22 RBS-lysis1-(1)	EcoRI	SpeI
2	8/22 RBS-lysis1-(1)	--	--
3	8/17 RBS-GFP-DT-(2)	XbaI	PstI
4	8/17 RBS-GFP-DT-(2)	--	--
5	8/20 Pcon-RBS-GFP-DT-(1)	EcoRI	SpeI
6	8/20 Pcon-RBS-GFP-DT-(1)	--	--
7	100bp ladder	--	--
8	8/20 Pcon-RBS-luxR-DT-(1)	EcoRI	XbaI
9	8/20 Pcon-RBS-luxR-DT-(1)	--	--
10	8/28 Plux	SpeI	PstI
11	8/28 Plux	--	--
12	8/20 RBS-tetR-DT	XbaI	PstI
13	8/20 RBS-tetR-DT	--	--

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	8/20 RBS-lysis1-(1)	EcoRI & SpeI
3	8/20 RBS-lysis1-(1)	EcoRI & SpeI
4	--	--
5	8/28 RBS-GFP-DT-(2)	XbaI & PstI
6	8/28 RBS-GFP-DT-(2)	XbaI & PstI

Lane	DNA	Enzyme
1	100bp ladder	--
2	8/28 Plux	SpeI & PstI
3	8/28 Plux	SpeI & PstI
4	--	--
5	8/20 RBS-tetR-DT-(1)	XbaI & PstI
6	8/20 RBS-tetR-DT-(1)	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
Pcon-RBS-GFP-DT (EcoRI&SpeI)	7.1	1.88	0.07
Pcon-RBS-luxR-DT (EcoRI&XbaI)	15.3	1.93	0.70
Plux (SpeI&PstI)	4.5	1.79	0.31
RBS-GFP-DT (XbaI&PstI)	6.0	2.16	0.03
RBS-tetR-DT (XbaI&PstI)	7.6	1.91	0.38

Colony PCR

No name

Sample	base pair
8/27 pT181 attenuator-DT-1	738
8/27 pT181 attenuator-DT-2	738
8/27 pT181 attenuator-DT-3	738
8/27 TetR-aptamer 12_1R-DT-1	521
8/27 TetR-aptamer 12_1R-DT-2	521
8/27 pT181 antisense-DT-1	542

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

Sample	base pair
8/27 Pλ-luxI-1	--
8/27 Pλ-luxI	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30min	3cycles

Colony PCR

No name

Sample	base pair
8/21 RBS-lysis2-9	772
8/21 RBS-lysis2-10	772
8/21 RBS-lysis2-11	772
8/21 RBS-lysis2-12	772
8/21 RBS-lysis2-13	772
8/21 RBS-lysis2-14	772

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	48s	30cycles

Liquid Culture

No name

Sample	medium
8/16 Master Plate(CP)-6 DT	Plusgrow medium(+CP)
J23100 Master Plate-1	Plusgrow medium(+Amp)
Plac Master Plate-1	Plusgrow medium(+CP)

Ligation

No name

state	Vector		Inserter
experiment	8/27 DT (EcoRI & XbaI)	2.9	8/28 RBS-lysis1 (EcoRI & SpeI)	9.5
experiment	8/28 Plux (SpeI & PstI)	2.3	8/28 RBS-GFP-DT (XbaI & PstI)	18
experiment	8/28 Pcon-RBS-luxR-DT (EcoRI & XbaI)	3.3	8/28 Pcon^RBS-GFP-DT-(1) (EcoRI & SpeI)	28
experiment	8/27 DT (EcoRI & XbaI)	2.7	8/24 Spinach (EcoRI & SpeI)	2.4

Samples were evaporeted used evaporator into about 1 µL.

sample	MilliQ	Ligation High	total
1	3	4	9

incubate overnight at 4 °C

Liquid Culture

No name

Sample	medium
8/26 Pλ-RBS-luxI-DT-1	Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2	Plusgrow medium (+Amp)

incubate 37°C 10hour

Aug 29

Colony PCR

No name

Sample	base pair
8/27 Pcon-pT181 antisense-(1)	542
8/26 pT181 attenuator-DT-(1)	738

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

Lane	Sample
1	100bp ladder
2	8/27 Pcon-pT181antisense-1
3	8/26 pT181attenuator-DT-1
4	100bp ladder

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	8/21 RBS-lysis2-9(Colony PCR prodution)
3	8/21 RBS-lysis2-8(Colony PCR prodution)
4	8/21 RBS-lysis2-7(Colony PCR prodution)
5	8/21 RBS-lysis2-10(Colony PCR prodution)
6	8/21 RBS-lysis2-11(Colony PCR prodution)
7	8/21 RBS-lysis2-12(Colony PCR prodution)
8	8/21 RBS-lysis2-13(Colony PCR prodution)
9	8/21 RBS-lysis2-14(Colony PCR prodution)

Miniprep

No Name

DNA	concentration [µg/mL]	260/280	260/230
Plac	153.7	2.03	2.10
Pλ-luxⅠ-(1)	76.5	2.07	2.06
Pλ-luxⅠ-(2)	86.4	2.08	2.10
DT	166.2	1.65	2.12
J23100	190.1	1.68	2.34

Liquid Culture

No name

Sample	medium
8/21 RBS-Lysis2-9	Plusgrow medium(+Amp)
8/21 RBS-Lysis2-12	Plusgrow medium(+Amp)
8/21 RBS-Lysis2-14	Plusgrow medium(+Amp)

Restriction Enzyme Digestion

No name

	8/22 psB1C3-(2)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts	8µL	0.5µL	0.5µL	0µL	0µL	3µL	0µL	0.3µL	17.7µL	30µL
NC	1µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	7.9µL	10µL
2 cuts	8µL	0µL	0µL	0.5µL	0.5µL	0µL	3µL	0.3µL	17.7µL	30µL
NC	1µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	8/21 tRMA-spinach(1)	XbaI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	4µL	0.5µL	0.5µL	3µL	0.3µL	21.7µL	30µL
NC	0.5µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	8/21 tetR aptamer12-1R(1)	EcoRI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	3µL	0.5µL	0.5µL	3µL	0.3µL	22.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	0.1µL	8.5µL	10µL

	8/21 PT181 attenuator-(2)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts	4µL	0.5µL	0.5µL	0µL	0µL	3µL	0µL	0.3µL	21.7µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.4µL	10µL
2 cuts	4µL	0µL	0µL	0.5µL	0.5µL	0µL	3µL	0.3µL	21.7µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	8/21 PT181 antisense-(2)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts	10µL	0.5µL	0.5µL	0µL	0µL	3µL	0µL	0.3µL	15.7µL	30µL
NC	1µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	7.9µL	10µL
2 cuts	10µL	0µL	0µL	0.5µL	0.5µL	0µL	3µL	0.3µL	15.7µL	30µL
NC	1µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

incubated 37°C 1hour

Transformation

No Name

Name	Sample	Competent Cells	Total	Plate
8/27 pT181 attenuator-DT	2µL	20µL	22µL	CP
8/27 RBS-lysis3-DT	2µL	20µL	22µL	CP
8/28 RBS-lysis3-DT	2µL	20µL	22µL	CP
8/28 Pcon-RBS-GFP-DT-Pcon-RBS-LuxR-DT	2µL	20µL	22µL	Amp
8/28 RBS-lysis1-DT	2µL	20µL	22µL	CP
8/28 Plux-RBS-GFP-DT	2µL	20µL	22µL	CP
8/28 Spinach-DT	2µL	20µL	22µL	CP

Gel Extraction

No name

Lane	DNA	Enzyme1	Enzyme2
	100bp ladder	--	--
9	8/29 pT181attenuator(2)	EcoRI	SpeI
10	8/29 pT181attenuator(2)	EcoRI	SpeI
11	8/29 pT181attenuator(2)	XbaI	PstI
12	8/29 pT181attenuator(2)	XbaI	PstI
13	pT181antisense(2)	EcoRI	SpeI
14	pT181antisense(2)	EcoRI	SpeI
15	pT181antisense(2)	XbaI	PstI
16	pT181antisense(2)	XbaI	PstI
	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
	100bp ladder	--	--
1	8/29 pSB1C3	EcoRI	SpeI
2	8/29 pSB1C3	EcoRI	SpeI
3	8/29 pSB1C3	XbaI	PstI
4	8/29 pSB1C3	XbaI	PstI
5	8/29 spinach	EcoRI	SpeI
6	8/29 spinach	EcoRI	SpeI
7	8/29 tetRaptarmer12-1R(1)	EcoRI	SpeI
8	8/29 tetRaptarmer12-1R(1)	EcoRI	SpeI
	100bp ladder	--	--

Aug 30

Gel Extraction

No Name

Name	quantity	concentration[µg/mL]	260/280	260/230
pT181 attenuator(2) (EcoRI & SpeI)	305.3	2.2	1.66	0.36
pT181 attenuator(2) (XbaI & PstI)	217.7	3.9	1.73	0.04
pT181 antisense(2) (EcoRI & SpeI)	347.7	3.1	1.88	0.31
pT181 antisense(2) (XbaI & PstI)	376.5	5.9	0.49	2.36
pSB1C3 (EcoRI & SpeI)	436.6	30.7	1.03	1.28
pSB1C3 (XbaI & PstI)	457.4	7.9	1.65	0.42
Spinach (EcoRI & SpeI)	490.5	3.0	1.87	0.26
tetR aptamer 12_1R (EcoRI & SpeI)	451.7	-12.8	5.56	-0.04

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
RBS-lysis2 9	264.6	1.97	2.21
RBS-lysis2 12	220.0	1.89	2.46
RBS-lysis2 14	195.3	1.88	1.53

Colony PCR

No name

Sample	base pair
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(8/20 LB Amp) (1)~(4)	2143
8/29 Plux-RBS-GFP-DT(8/27 LB-CP) (1)~(2)	1227
8/29 Pbad/araC-RBS-RFP{BBa_I13516} (8/21 LB-CP) (1)~(2)	2256
8/29 RBS-lysis3-DT-(1)	1210

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycle

Ethanol Precipitation

No name

DNA
pT181 attenuator-(2) (EcoRI & SpeI)
pT181 attenuator-(2) (XbaI & PstI)
pT181 antisense-(2) (EcoRI & SpeI)
pT181 antisense-(2) (XbaI & PstI)
pSB1C3 (EcoRI & SpeI)
pSB1C3 (XbaI & PstI)
Spinach (EcoRI & SpeI)
tetR aptamer 12_1R (EcoRI & SpeI)

Restriction Enzyme Digestion

No name

	8/30 RBS-lysis2 (9)	EcoRI	SpeI	10x bufferB	100x BSA	MilliQ	total
2 cuts	7.5 µL	0.5 µL	0.5 µL	3 µL	0.3 µL	18.2 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	0.1 µL	8.4 µL	10 µL

	8/22 pSB1C3-(2)	EcoRI	SpeI	10x Buffer	100x BSA	MilliQ	total
2 cuts	8 µL	0.5 µL	0.5 µL	3 µL	0.3 µL	17.7 µL	30 µL
NC	1 µL	0 µL	0 µL	1 µL	0.1 µL	7.9 µL	10 µL

	8/22 pSB1C3-(2)	XbaI	PstI	BufferD	BSA	MilliQ	total
2 cuts	8 µL	0.5 µL	0.5 µL	3 µL	0.3 µL	17.7 µL	30 µL
NC	1 µL	0 µL	0 µL	1 µL	0.1 µL	7.9 µL	10 µL

	8/21 Spinach (1)	EcoRI	SpeI	10x BufferB	100x BSA	MilliQ	total
2 cuts	4 µL	0.5 µL	0.5 µL	3 µL	0.3 µL	21.9 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	0.1 µL	8.4 µL	10 µL

	8/21 tetR aptamer 12_1R (1)	EcoRI	SpeI	10xBufferB	100xBSA	MilliQ	total
2 cuts	3 µL	0.5 µL	0.5 µL	3 µL	0.3 µL	22.7 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	0.1 µL	8.4 µL	10 µL

	8/21 pT181 attenuator-(2)	EcoRI	SpeI	XbaI	PstI	10x BufferB	10x BufferD	100x BSA	MilliQ	total
2 cuts	4 µL	0.5 µL	0.5 µL	0 µL	0 µL	3 µL	0 µL	0.3 µL	21.7 µL	30 µL
NC	0.5 µL	0 µL	0 µL	0 µL	0 µL	1 µL	0 µL	0.1 µL	8.4 µL	10 µL
2 cuts	4 µL	0 µL	0 µL	0.5 µL	0.5 µL	0 µL	3 µL	0.3 µL	21.7 µL	30 µL
NC	0.5 µL	0 µL	0 µL	0 µL	0 µL	0 µL	1 µL	0.1 µL	8.4 µL	10 µL

	8/21 pT181 antisense-(2)	EcoRI	SpeI	XbaI	PstI	10x BufferB	10x BufferD	100x BSA	MilliQ	total
2 cuts	9 µL	0.5 µL	0.5 µL	0 µL	0 µL	3 µL	0 µL	0.3 µL	16.7 µL	30 µL
NC	1 µL	0 µL	0 µL	0 µL	0 µL	1 µL	0 µL	0.1 µL	7.9 µL	10 µL
2 cuts	9 µL	0 µL	0 µL	0.5 µL	0.5 µL	0 µL	3 µL	0.3 µL	16.7 µL	30 µL
NC	1 µL	0 µL	0 µL	0 µL	0 µL	0 µL	1 µL	0.1 µL	7.9 µL	10 µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	RBS-lysis2-(9)	EcoRI	SpeI
3	RBS-lysis2-(9)	--	--
4	pSB1C3-(2)	EcoRI	SpeI
5	pSB1C3-(2)	--	--
6	pSB1C3-(2)	XbaI	PstI
7	pSB1C3-(2)	--	--
8	tRNA-Spinach-(1)	EcoRI	SpeI
9	tRNA-Spinach-(1)	--	--
10	tetR aptamer12_1R	EcoRI	SpeI
11	tetR aptamer12_1R	--	--
12	pT181 attenuator	EcoRI	SpeI
13	pT181 attenuator	EcoRI	SpeI
14	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	pT181 antisense	EcoRI	SpeI
3	pT181 antisense	--	--
4	pT181 antisense	XbaI	PstI
5	pT181 antisense	--	--
6	pT181 attenuator	XbaI	PstI
7	pT181 attenuator	--	--
8	100bp ladder	--	--

Lane	Sample
1	1kb ladder
2	8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)(Colony PCR product)
3	8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)(Colony PCR product)
4	8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)(Colony PCR product)
5	8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)(Colony PCR product)
6	Pbad/araC-RBS-RFP-DT-(1)(Colony PCR product)
7	Pbad-araC-RBS-RFP-DT-(2)(Colony PCR product)

Liquid Culture

No name

Sample	medium
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)	Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)	Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)	Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)	Plusgrow medium(+Amp)
8/29 Plux-RBS-GFP-DT-(1)	Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT-(2)	Plusgrow medium(+CP)
8/29 Pbad/araC-RBS-RFP-DT-(1)	Plusgrow medium(+CP)
8/29 Pbad-araC-RBS-RFP-DT-(2)	Plusgrow medium(+CP)
RBS-lysis3-DT-(1)	Plusgrow medium(+CP)

Aug 31

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	--	--
3	RBS-lysis2-(9)	EcoRI & SpeI
4	RBS-lysis2-(9)	EcoRI & SpeI
5	--	--
6	pSB1C3(1)	EcoRI & SpeI
7	pSB1C3(1)	EcoRI & SpeI
8	--	--
9	pSB1C3(1)	XbaI & PstI
10	pSB1C3(1)	XbaI & PstI

Lane	DNA	Enzyme
1	100bp ladder	--
2	tRNA-Spinach	EcoRI & SpeI
3	tRNA-Spinach	EcoRI & SpeI
4	--	--
5	pT181 attenuator-(2)	EcoRI & SpeI
6	pT181 attenuator-(2)	EcoRI & SpeI
7	pT181 attenuator-(2)	XbaI & PstI
8	pT181 attenuator-(2)	XbaI & PstI
9	--	--
10	pT181 antisense-(2)	EcoRI & SpeI
11	pT181 antisense-(2)	EcoRI & SpeI

Lane	DNA	Enzyme
1	100bp ladder	--
2	pT181 antisense	XbaRI & PstI
3	pT181 antisense	XbaRI & PstI
4	--	--
5	tetR aptamer 12_1R-(1)	EcoRI & SpeI
6	tetR aptamer 12_1R-(1)	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis2(EcoRI&SpeI)	4.0	2.69	0.24
pSB1C3(EcoRI&SpeI)	6.0	2.48	0.05
pSB1C3(XbaI&PstI)	--	--	--
tRNA-Spinach	--	--	--
pT181 attenuator-(2)(EcoRI&SpeI)	--	--	--
pT181 anttenuator-(2)(SbaI&PstI)	--	--	--
pT181 antisense-(2)(EcoRI&SpeI)	--	--	--
pT181 antisense-(2)(XbaI&PstI)	--	--	--
tetR aptamer 12_1R(EcoRI&SpeI)	--	--	--

Ligation

Nakamoto and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	8/30 pSB1C3 (EcoRI & SpeI)	6.6	8/30 tRNA-Spinach (EcoRI & SpeI)	12.5	3.5
experiment	8/30 pSB1C3 (EcoRI & SpeI)	6.6	8/30 pT181 antisense (EcoRI & SpeI)	12.5	3.5
experiment	8/30 pSB1C3 (XbaI & Pst)	12.5	8/30 pT181 attenuator (XbaI & PstI)	25	3.5
experiment	8/30 pSB1C3 (XbaI & Pst)	12.5	8/30 pT181 antisense (XbaI & PstI)	25	3.5
experiment	8/20 DT (EcoRI & XbaI)	6.6	8/30 pT181 attenuator(2) (EcoRI & SpeI)	12.5	3.5
experiment	8/20 DT (EcoRI & XbaI)	6.6	8/30 pT181 antisense (EcoRI & SpeI)	12.5	3.5
experiment	8/27 DT (EcoRI & XbaI)	3.0	8/30 tRNA-Spinach (EcoRI & SpeI)	12.5	3.5
experiment	8/20 DT (EcoRI & XbaI)	6.6	8/30 RBS-lysis2 (EcoRI & SpeI)	12.5	3.5
experiment	8/22 J23100 (SpeI & PstI)	--	RBS-tetR-DT (XbaI & PstI)	--	3.5
experiment	8/22 Plac (SpeI & PstI)	8.0	8/18 RBS-lacZα-DT (XbaI & PstI)	10	3.5

Samples were evaporeted used evaporator into about 7 µL

Miniprep

Kojima and Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
8/30 Plux-RBS-GFP-DT-(1)	124	1.77	1.87
8/30 Plux-RBS-GFP-DT-(2)	213	1.75	1.52
8/30 Pbad/araC-RBS-RFP	427	1.70	1.56
8/30 Pbad/araC-RBS-RFP	670	1.30	0.83
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)	332	1.73	2.04
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)	478	1.74	2.09
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)	146	1.77	2.11
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)	454	1.75	2.19

Sep 1

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
8/31 tRNA-Spinach(pSB1C3)	2µL	20µL	22µL	CP
8/31 pT181 antisense(pSB1C3)	2µL	20µL	22µL	CP
8/31 pT181 attenuator(pSB1C3)	2µL	20µL	22µL	CP
8/31 pT181 antisense(pSB1C3)	2µL	20µL	22µL	CP
8/31 pT181 attenuator(2)-DT	2µL	20µL	22µL	CP
8/31 pT181 antisense-DT	2µL	20µL	22µL	CP
8/31 tRNA-Spniach-DT	2µL	20µL	22µL	CP
8/31 RBS-lysis2-DT	2µL	20µL	22µL	CP
8/31 Pconst-RBS-tetR-DT	2µL	20µL	22µL	Amp
8/31 Plac-RBS-lacZα-DT	2µL	20µL	22µL	CP
8/31 Ptet-RBS-lacZα-DT	2µL	20µL	22µL	CP
8/31	2µL	20µL	22µL	CP

ligation production PCR

No name

Sample	base pair
8/31 tRNA Spinach(pSB1C3)	459
8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI)	415
8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI)	601
8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI)	415
8/31 pT181 attenuator(2)-DT	754
8/31 pT181 antisense(2)-DT	577
8/31 tRNA Spinach-DT	596
8/22 pSB1C3(2)(controll)	1383

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30min	30cycle

Ligation

Nakamoto

state	Vector		Inserter
experiment	DT(EcoRI+SpeI)	5.5	8/28 RBS-lysis1(EcoRI+SpeI)2.4ng/µL	2.4

1. Samples were evaporeted used evaporator into about 7 µL.
2. Add Ligation High 3.5&micro:L and incubate 16°C 1hour.

Liquid Culture

Tatsui,Stephane,Nakamoto

Sample	medium
8/21 pSB1C3(BBa_J04450)-(1)	Plusgrow medium(+CP)
8/21 pSB1C3(BBa_J04450)-(2)	Plusgrow medium(+CP)
RBS-lysis3-DT	Plusgrow medium(+CP)

M9 Medium

No name 5xM9 liquid medium

volume	100mL
NaHPO4	30g
KH2PO	3g
Nacl	0.25g
NH4Cl	0.5g
3.75% agar solution	400mL
1M MgSO4	0.5mL
2M Glucose	2.8mL
1% Vitamin B1	0.5mL
1M CaCl2	0.05mL

1. mix 5xM9 medium and 3.75%Agar solution 400mL
2. Add 1M MgSO4, 2M Glucose and 1 VitaminB1 0.5mL
3. Add CaCl2 while shaking Erlenmeyer flask.

Miniprep

DNA	concentration[µg/mL]	260/280	260/230
8/21 pSB1C3(1)	309.2	1.69	1.72
8/21 pSB1C3(2)	201.6	1.65	1.76

Kanamycin applicaton

Hirano

Add 20µL Kanamycin into 80µL MilliQ and apply it on a 9/1 M9 plate

Plating

Hirano

Apply entA-colony liquid culture (100µL) on M9(Km)plate and incubate 37°C

2 fold diluted OD 600 2.107

Liquid Culture

Hirano

Sample	medium
9/1 entA- Master Plate-(4)	SOB Medium(+Km)

Sep 2

Colony PCR

Tatsui

Sample	base pair
9/1 RBS-lysis2-DT-(1)	985
Ptet-RBS-lacZα-DT-(1)	765
Plac-RBS-lacZα-DT-(1)	765
Plac-RBS-lacZα-DT-(2)	765

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	9/1 RBS-lysis2+DT -(1)
3	9/1 Ptet+RBS-lacZα-DT -(1)
4	9/1 Plac+RBS-lacZα-DT -(1)
5	9/1 Plac+RBS-lacZα-DT -(2)
6	100bp ladder

Restriction Enzyme Digestion

Nakamoto

	pSB1C3-(1)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts(EcoRI+SpeI)	7µL	1µL	1µL	0µL	0µL	3µL	0µL	0.3µL	17.7µL	30µL
NC(EcoRI+SpeI)	0.3µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.6µL	10µL
2 cuts(XbaI+PstI)	7µL	0µL	0µL	1µL	1µL	0µL	3µL	0.3µL	17.7µL	30µL
NC(XbaI+PstI)	0.3µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

	8/21 tRMA-spinach(1)	EcoRI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	8µL	1.0µL	1.0µL	3µL	0.3µL	17.6µL	30µL
NC	0.5µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	8/21 tetR aptamer12_1R-(1)	EcoRI	SpeI	BufferB	BSA	MilliQ	total
2 cuts	6µL	1.0µL	1.0µL	3µL	0.3µL	18.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	0.1µL	8.5µL	10µL

	8/21 pT181 attenuator-(2)	EcoRI	SpeI	XbaI	PstI	BufferB	BufferD	BSA	MilliQ	total
2 cuts(EcoRI+SpeI)	8µL	1µL	1µL	0µL	0µL	3µL	0µL	0.3µL	16.3µL	30µL
NC(EcoRI+SpeI)	0.5µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.4µL	10µL
2 cuts(XbaI+PstI)	8µL	0µL	0µL	1µL	1µL	0µL	3µL	0.3µL	16.3µL	30µL
NC(XbaI+PstI)	0.5µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

Liquid Culture

Kojima

Sample	medium
tRNA-Spinach-1	Plusgrow medium(+CP)
tetR aptamaer12_1R-1	Plusgrow medium(+CP)
pT181 attenuator-1	Plusgrow medium(+CP)
pT181 antisense-1	Plusgrow medium(+CP)

Master Plate

Kojima

Number	Use LB plate(+CP)
1	tRNA Spinach-1
2	tetR aptamaer12_1R
3	tetR aptamaer12_P
4	tetR aptamaer12_1M
5	pT181 attenuator-2
6	Fusion1 attenuator-1
7	Fusion3m2 attenuator-1
8	pT181 antisense-1
9	Fusion1 antisense-1
10	Fuaion6 antisense-1

Electrophoresis

Nakamoto & Tatsui

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	pSB1C3 -(1)	EcoRI	SpeI
3	pSB1C3 -(1)	--	--
5	pSB1C3 -(1)	XbaI	PstI
6	pSB1C3 -(1)	--	--
7	pT181 attenuator (2)	EcoRI	SpeI
8	pT181 attenuator (2)	--	--
9	pT181 attenuator (2)	XbaI	SpeI
10	pT181 attenuator (2)	--	--
11	tetR-aptamer 12_1R	EcoRI	SpeI
12	tetR-aptamer 12_1R	--	--
13	tRNA Spinach (1)	EcoRI	SpeI
14	100bp ladder	--	--
15	tRNA Spinach (1)	--	--

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	-
2	pSB1C3(EcoRI+SpeI)	EcoRI&SpeI
3	pSB1C3(EcoRI+SpeI)	EcoRI&SpeI
4	--	--
5	8/24 tetR aptamer 12_1R	EcoRI&SpeI
6	8/24 tetR aptamer 12_1R	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder	-
2	pT181 attenuator(2)(EcoRI+SpeI)	EcoRI&SpeI
3	pT181 attenuator(2)(EcoRI+SpeI)	EcoRI&SpeI
4	--	--
5	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
6	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
7	--	--
8	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
9	pT181 attenuator(2)(XbaI+PstI)	EcoRI&SpeI
10	--	--
11	Spinach	EcoRI&SpaI
12	Spinach	EcoRI&SpaI

Name	concentration[µg/mL]	260/280	260/230
pSB1C3 (EcoRI&SpeI)	3.0	2.18	0.31
pSB1C3 (XbaI&PstI)	4.7	2.25	0.36
pT181 attenuator-(2)(EcoR&SpeI)	8.6	2.74	0.01
pT181 attenuator-(2) (XbaI&PstI)	16.5	2.46	0.03
Spinach (EcoRI&SpeI)	2.8	2.98	0.27
tetR aptamer12_1R (EcoRI&SpeI)	50.4	28.07	1.97

Colony PCR

No name

Sample	base pair
9/1 Spinach(pSB1C3)	about 450
9/1 Spinach-DT	596
9/1 RBS-lysis1-DT	613

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	40s	30cycles

Sample	base pair
9/1 Ptet-RBS-lacZα-DT-2	738
9/1 Ptet-RBS-lacZα-DT-3	738
9/1 Ptet-RBS-lacZα-DT-4	738
9/1 Ptet-RBS-lacZα-DT-5	738
9/1 Ptet-RBS-lacZα-DT-6	738
9/1 RBS-lysis2-DT-2	985
9/1 RBS-lysis2-DT-3	985

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min	30cycles

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	Spinach(pSB1C3)
3	Spinach-DT
4	RBS-lysis1-DT
5	100bp ladder

Master Plate

No name

Number	Use LB plate(+CP)
1	9/1 Spinach(pSB1C3)
2	9/1 Spinach-DT-(1)

Liquid Culture

No name

Sample	medium
pT181 attenuator	Plusgrow medium (+CP)
pT181 antisense	Plusgrow medium (+CP)
Spinach	Plusgrow medium (+CP)

incubated at 37 °C for 1 hour

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
8/28 RBS-lysis3-DT	3µL	30µL	33µL	CP

Sep 3

Electrophoresis

Kojima

Lane	Sample
1	100bp ladder
2	pT181 attenuator(2)-DT2(738)
3	Ptet-RBS-lacZα-DT-2(756)
4	Ptet-RBS-lacZα-DT-3
5	Ptet-RBS-lacZα-DT-4
6	Ptet-RBS-lacZα-DT-5
7	Ptet-RBS-lacZα-DT-6
8	RBS-lysis2-DT-2(985)
9	RBS-lysis2-DT-3
10	100bp ladder

Miniprep

Tatsui

DNA	concentration[µg/mL]	260/280	260/230
9/2 14:00 pT181 attenuator	418.4	1.82	1.80
9/2 14:00 pT181 antisense	362.2	1.92	1.96
9/2 14:00 Spinach	438.3	1.86	1.99
9/2 14:00 tetR-apt 12_1R	567.5	1.90	1.99
9/2 21:00 pT181 attenuator	320.9	1.90	2.15
9/2 21:00 Spinach	457.0	1.88	2.24

Liquid Culture

Kojima

Sample	medium
Spinach-DT(9/2 master plate)	plusgrow(CP)4ml

Restriction Enzyme Digestion

Kojima

	DNA	EcoRI	SpeI	XbaI	PstI	10xBuffer B(EcoRI&SpeI)	10xBuffer D(XbaI&PstI)	100xBSA	MilliQ	total
9/3 pT181attenuator 14:00 418.4ng/µL	4.8µL	1.0µL	1.0µL	0µL	0µL	3µL	0µL	0.3µL	19.9µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.2µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.7µL	10µL
9/3 pT181attenuator 14:00 418.4ng/µL	4.8µL	0µL	0µL	1.0µL	1.0µL	0µL	3µL	0.3µL	19.9µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.2µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.7µL	10µL

	DNA	EcoRI	SpeI	XbaI	PstI	10xBuffer B(EcoRI&SpeI)	10xBuffer D(XbaI&PstI)	100xBSA	MilliQ	total
9/3 pT181antisense 14:00 362.2ng/µL	5.5µL	1.0µL	1.0µL	0µL	0µL	3µL	0µL	0.3µL	19.2µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.3µL	0µL	0µL	0µL	0µL	1µL	0µL	0.1µL	8.6µL	10µL
9/3 pT181attenuator 14:00 418.4ng/µL	5.5µL	0µL	0µL	1.0µL	1.0µL	0µL	3µL	0.3µL	19.2µL	30µL
9/3 pT181attenuator 14:00 418.4ng/µL	0.3µL	0µL	0µL	0µL	0µL	0µL	1µL	0.1µL	8.6µL	10µL

	DNA	EcoRI	SpeI	10xBuffer B	100xBSA	MilliQ	total
9/3 apt 12_1R 14:00 507.5ng/µL	3.9µL	1.0µL	1.0µL	3µL	0.3µL	20.8µL	30µL
9/3 apt 12_1R 14:00 507.5ng/µL	0.2µL	0µL	0µL	1µL	0.1µL	8.7µL	10µL

	DNA	EcoRI	XbaI	10xBuffer D	100xBSA	MilliQ	total
8/29 DT 166.2ng/µL	12µL	1.0µL	1.0µL	3µL	0.3µL	12.7µL	30µL
8/29 DT 166.2ng/µL	0.6µL	0µL	0µL	1µL	0.1µL	8.3µL	10µL

	DNA	EcoRI	SpeI	10xBuffer B	100xBSA	MilliQ	total
8/22 RBS-lysis1(2) 96ng/µL	21µL	1.0µL	1.0µL	3µL	0.3µL	3.7µL	30µL
8/22 RBS-lysis1(2) 96ng/µL	1.0µL	0µL	0µL	1µL	0.1µL	7.9µL	10µL

	DNA	EcoRI	SpeI	10xBuffer B	100xBSA	MilliQ	total
8/30 RBS-lysis2 220ng/µL	9.1µL	1.0µL	1.0µL	3µL	0.3µL	15.6µL	30µL
8/30 RBS-lysis2 220ng/µL	0.5µL	0µL	0µL	1µL	0.1µL	8.4µL	10µL

	DNA	EcoRI	SpeI	10xBuffer B	100xBSA	MilliQ	total
8/20 RBS-lysis3(1) 282ng/µL	7.1µL	1.0µL	1.0µL	3µL	0.3µL	17.6µL	30µL
8/20 RBS-lysis3(1) 282ng/µL	0.4µL	0µL	0µL	1µL	0.1µL	8.5µL	10µL

PCR

Hirano

Plac(1) plasmid 153.7ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer (KaiA_BsaI_Mut_fwd)	primer (KaiA_BsaI_Mut_rer)	KOD plus-ver.2	MilliQ	total
0.13	2.5	2.5	1.5	0.75	0.75	0.5	16.37	25

KaiB (plasmid) 20.2ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer (GG2_RBS_KaiB_fwd)	primer (KaiB_GG3_rer)	KOD plus-ver.2	MilliQ	total
1	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

KaiC (plasmid) 4.4ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
1	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

DT-1 (plasmid) 153.7ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.1	2.5	2.5	1.5	0.75	0.75	0.5	16.4	25

PKaiBC(PCR product) 16.3ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.7	2.5	2.5	1.5	0.75	0.75	0.5	15.8	25

SasA(PCR product) 21.6ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.5	2.5	2.5	1.5	0.75	0.75	0.5	16	25

RpaA(PCR product) 15.4ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.6	2.5	2.5	1.5	0.75	0.75	0.5	15.9	25

RpaB(PCR product) 26.7ng/µL	10x buffer KOD plus-ver.2	2mM dNTPs	25mM MgSO4	primer	primer	KOD plus-ver.2	MilliQ	total
0.4	2.5	2.5	1.5	0.75	0.75	0.5	16.1	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C		68°C	--
5min	10sec	30sec		30

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C		68°C	--
5min	10sec	30sec		30

Mutatation PCR

No name

KaiABC(12.6pg/µL)	Primer(Kai_BsaIMut_fwd)	Primer(Kai_BsaIMut_rer)	Prime STAR Max Premix	MilliQ	total
1	1	1	25	22	50

	Primer(Kai_BsaIMut_fwd)	Primer(Kai_BsaIMut_rer)	Prime STAR Max Premix	MilliQ	total
	1	1	25	23	50

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	65°C	72°C	--
5min	10sec	15sec	40sec	30

Colony PCR

Sample	base pair
9/1 pT181antisense(pSB1C3)	400bp
9/1 pT181antisense-DT	547bp

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5m	30s	30s	30s	30

Electrophoresis

Kojima

Lane	Sample
1	100bp ladder
2	9/1 pT181antisense(pSB1C3)
3	9/1 pT181antisense

Electrophoresis

Lane	Sample
1	100bp ladder
2	attenuator EcoRI&SpeI
3	attenuator negative EcoRI&SpeI
4	attenuator XbaI&PstI
5	attenuator negative XbaI&PstI
6	antisense EcoRI&SpeI
7	antisense negative EcoRI&SpeI
8	100bp ladder

Lane	Sample
1	100bp ladder
2	antisense XbaI&PstI
3	antisense negative XbaI&PstI
4	apt EcoRI&SpeI
5	apt negative EcoRI&SpeI
6	DT EcoRI&XbaI
7	DT negative EcoRI&XbaI
8	100bp ladder

Lane	Sample
1	100bp ladder
2	RBS-lysis1 EcoRI&SpeI
3	RBS-lysis1 negative
4	RBS-lysis2 EcoRI&SpeI
5	RBS-lysis2 negative
6	RBS-lysis3 EcoRI&SpeI
7	RBS-lysis3 negative
8	100bp ladder

Gel Extraction

Lane	DNA	Enzyme
1	100bp ladder
3	pT181 attenuator	EcoRI&SpeI
4	pT181 attenuator	EcoRI&SpeI
6	pT181 attenuator	XbaI&PstI
7	pT181 attenuator	XbaI&PstI
9	pT181 antisense	EcoRI&SpeI
10	pT181 antisense	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder
2	pT181 antisense	XbaI&PstI
3	pT181 antisense	XbaI&PstI
5	apt12_1R	EcoRI&SpeI
6	apt12_1R	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder
2	DT	EcoRI&XbaI
3	DT	EcoRI&XbaI
5	RBS-lysis1	EcoRI&SpeI
6	RBS-lysis1	EcoRI&SpeI

Lane	DNA	Enzyme
1	100bp ladder
2	RBS-lysis2	EcoRI&SpeI
3	RBS-lysis2	EcoRI&SpeI
5	RBS-lysis3	EcoRI&SpeI
6	RBS-lysis3	EcoRI&SpeI

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator(EcoRI&SpeI)	10.0	1.86	0.39
pT181 attenuator(XbaI&PstI)	11.7	1.65	0.36
pT181 antisense(EcoRI&SpeI)	4.2	1.95	0.25
pT181 antisense(XbaI&PstI)	6.6	1.78	0.33
tRNA 12_1R apt(EcoRI&SpeI)	5.7	1.73	0.29
DT(EcoRI&XbaI)	16.6	1.99	0.76
RBS-lysis1(EcoRI&SpeI)	8.8	1.65	0.32
RBS-lysis2(EcoRI&SpeI)	13.4	1.61	0.48
RBS-lysis3(EcoRI&SpeI)	10.2	2.19	0.51

Liquid Culture

No name

Sample	medium
8/16master plate7(DT)
8/21RBS-lysis1

Sep 4

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/3 DT(EcoRI&XbaI)	3.2µL	9/3 RBS-lysis1	2.4µL	2.7µL
experiment	9/3 DT(EcoRI&XbaI)	3.0µL	9/3 RBS-lysis2	4.7µL	4.0µL
experiment	9/3 DT(EcoRI&XbaI)	3.0µL	9/3 RBS-lysis3	8.8µL	4.0µL
experiment	9/3 DT(EcoRI&XbaI)	3.0µL	9/3 aptamer12_1R(EcoRI&SpeI)	1.9µL	2.45µL
experiment	9/2 pSB1C3(XbaI&PstI)	10.6µL	9/3 pT181attenuator (XbaI& PstI)	3.2µL	4.0µL
experiment	9/2 pSB1C3(XbaI&PstI)	9.6µL	9/3 pT181antisense (XbaI& PstI)	2.1µL	4.0µL
experiment	9/2 pSB1C3(EcoRI&SpeI)	16.7µL	9/3 aptamer12_1R(EcoRI&SpeI)	2.0µL	4.0µL

incubate 16 °C 1 hour

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/4 RBS-lysis1-DT	2µL	20µL	Amp
9/4 RBS-lysis2-DT	2µL	20µL	Amp
9/4 RBS-lysis3-DT	2µL	20µL	Amp
9/4 aptamer12_1R-DT	2µL	20µL	Amp
9/4 pT181attenuator –pSB1C3	2µL	20µL	Amp
9/4 pT181antisense-pSB1C3	2µL	20µL	Amp
9/4 aptamer12_1R –pSB1C3	2µL	20µL	Amp
9/4 pSB4K5(2013plate5 56)	2µL	20µL	Amp
9/3 Mutagenesis product 1(KaiABC)	2µL	20µL	Amp
9/3 Mutagenesis product 2(NC)	2µL	20µL	Amp

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
DT	193.1	1.93	1.81
Spinach-DT	373.3	1.92	1.69
RBS-lysis1	332.0	1.95	1.62

Master Plate

Hirano

Number	Use LB plate (+CP)
1	8/10 Plac(CP)-2
2	8/10 Plac(CP)-2
3	8/10 Plac(CP)-2

Number	Use LB plate (+Amp)
1	8/9 J23100(Amp)-1
2	8/9 J23100(Amp)-1
3	8/9 J23100(Amp)-1

Liquid Culture

Hirano

Sample	medium
8/10 Plac(CP)-2	Plusgrow medium(+CP)
8/9 J23100(Amp)-1	Plusgrow medium(+Amp)

PCR

Hirano

8/29 Pλ-RBS-luxI-DT	KOD plus	10x buffer	dNTP	MgSO4	F primer (RBS-luxI-DT-cloning-fwd)	R primer(RBS-luxI-DT-cloning-rev)	MilliQ	total
0.3µL	0.5µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	16.2µL	25µL

8/31 Pbad/araC-RBS-RFP(1)	KOD plus	10x buffer	dNTP	MgSO4	F primer (Pbad/araC-pSB1C3-cloning-fond)	R primer(Pbad/araC-cloning-rex)	MilliQ	total
0.3µL	0.5µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	16.2µL	25µL

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	65°C	68°C	--
5min	10s	30s	3min10s	30cycles

Restriction Enzyme Digestion

No name

	8/21 Pconst	SpeI	PstI	Buffer	BSA	MilliQ	total
2 cuts	10.5µL	1.0µL	1.0µL	3.0µL	0.3µL	14.2µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	0.1µL	8.4µL	10 µL

	8/29 Plac	SpeI	PstI	Buffer	BSA	MilliQ	total
2 cuts	13.0µL	1.0µL	1.0µL	3.0µL	0.3µL	11.7µL	30 µL
NC	0.7µL	0µL	0µ	1.0µL	0.1µL	8.2µL	10 µL

	8/20 Ptet	SpeI	PstI	Buffer	BSA	MilliQ	total
2 cuts	9.0µL	1.0µL	1.0µL	3.0µL	0.3µL	15.7µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	0.1µL	8.4µL	10 µL

	8/20 RBS-tetR-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	8.5µL	1.0µL	1.0µL	3.0µL	0.3µL	16.2µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	0.1µL	8.4µL	10 µL

	spinach -DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.4µL	1.0µL	1.0µL	3.0µL	3.0µL	16.6µL	30 µL
NC	0.3µL	0µL	0µ	1.0µL	1.0µL	7.7µL	10 µL

	8/17 RBS-lacZα-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	11µL	1.0µL	1.0µL	3.0µL	3.0µL	11µL	30 µL
NC	0.5µL	0µL	0µ	1.0µL	1.0µL	7.5µL	10 µL

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
Ptrc KaiC	1µL	10µL	11µL	Amp
Ptrc KaiB	1µL	10µL	11µL	Amp
pSΩ	1µL	10µL	11µL	Amp

Electrophoresis

No name
1

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pconst	SpeI	PstI
3	Pconst NC	--	--
4	Plac	SpeI	PstI
5	Plac NC	--	--
6	100bp ladder	--	--

2

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Ptet	SpeI	PstI
3	Ptet NC	--	--
4	RBS-tetR-DT	XbaI	PstI
5	RBS-tetR-DT NC	--	--
6	100bp ladder	--	--

3

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Spinach-DT	XbaI	PstI
3	Spinach-DT NC	--	--
4	RBS-lacZα-DT	XbaI	PstI
5	RBS-lacα-DT NC	--	--
6	100bp ladder	--	--

Gel Extraction

No name

1

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pcon	SpeI&PstI
3	Pcon	SpeI&PstI
4	--	--
5	Plac	SpeI&PstI
6	Plac	SpeI&PstI

2

Lane	DNA	Enzyme
1	100bp ladder	--
2	Ptet	SpeI&PstI
3	Ptet	SpeI&PstI
4	--	--
5	RBS-tetR-DT	XbaI&PstI
6	RBS-tetR-DT	XbaI&PstI

Name	quantity	concentration[µg/mL]	260/280	260/230
Pcon	--	13.2	1.99	-1.64
Plac	--	15.3	2.07	-0.39
Ptet	--	30.1	1.97	-0.97
RBS-tetR-DT	--	3.0	2.44	0.88

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	spinach-DT	XbaI&PstI
3	spinach-DT	XbaI&PstI
4	--	--
5	RBS-lacZα-DT	XbaI&PstI
6	RBS-lacZα-DT	XbaI&PstI

Name	quantity	concentration[µg/mL]	260/280	260/230
spinach-DT	--	2.1	2.10	-0.05
RBS-lacZα-DT	--	3.3	2.59	-0.07

Master Plate

No name

Number	Use LB plate (+Amp)
4	8/9 BBa_J23100-3
5	8/9 BBa_J23100-4
6	8/9 BBa_J23100-5

Number	Use LB plate
1	JM109-1
2	JM109-2
3	JM109-3
4	JM109-4

Liquid Culture

Hirano

Sample	medium
8/9 BBa_J23100-3	Plusgrow medium(+Amp)
JM109-1(Master plate)	LB medium

Colony PCR

Hirano

Sample	base pair
8/9 BBa_J23100 3	--
8/9 BBa_J23100 4	--
8/9 BBa_J23100 5	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

Sep 5

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	J23100-3	--	--
3	J23100-4	--	--
4	J23100-5	--	--
5	100bp ladder	--	--

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
8/3 J23100-3	349.1	1.69	1.851
8/10 Plac-2	331.3	1.84	1.42

Ligation

Kojima

state	Vector		Inserter		Ligation High ver.2
experiment	Pconst	13.2	spinach-DT	2.1	3.5
experiment	Plac	15.3	spinach-DT	2.1	3.5
experiment	Pconst	13.2	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Plac	15.3	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Ptet	30.1	pT181 antisense (XbaI & PstI)	6.6	3.5
experiment	Pconst	13.2	pT181 attenuator	11.7	3.5
experiment	Plac	15.3	pT181 attenuator	11.7	3.5
experiment	Ptet	30.1	RBS-lacZα-DT	3.0	3.5

incubate 16 °C 1 hour

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	RBS-luxI-DT
3	Pbad/araC
4	NC
5	100bp ladder

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder
2	RBS-luxI-DT
3	RBS-luxI-DT
4
5	Pbad/araC
6	Pbad/araC

Name	concentration[µg/mL]	260/280	260/230
RBS-luxI-DT	23.2	0.83	7.89
Pbad/araC	51.8	1.20	0.11

Colony PCR

No name

Sample	base pair
9/4 aptamer 12_1R(pSB1C3)-1	384
9/4 aptamer 12_1R(pSB1C3)-2	384
9/4 pT181 antisense(pSB1C3)-1	415
9/4 pT181 antisense(pSB1C3)-2	415
9/4 pT181 antisense(pSB1C3)-3	415
9/4 pT181 antisense(pSB1C3)-4	415

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	30s	30cycles

Sample	base pair
9/4 aptamer 12_1R-DT-1	521
9/4 aptamer 12_1R-DT-2	521
9/4 aptamer 12_1R-DT-3	521
9/4 aptamer 12_1R-DT-4	521
9/4 pT181 attenuator(pSB1C3)	601
9/4 pT181 attenuator(pSB1C3)	601
9/4 RBS-lysis1-DT	613
9/4 RBS-lysis1-DT	613

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	36s	30cycles

Sample	base pair
9/4 RBS-lysis2-DT-1	985
9/4 RBS-lysis2-DT-2	985
9/4 RBS-lysis2-DT-3	985
9/4 RBS-lysis2-DT-4	985
9/4 RBS-lysis3-DT-1	1210
9/4 RBS-lysis3-DT-2	1210

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	72s	30cycles

Liquid Culture

Nakamoto

Sample	medium
8/9 J23100-5	Plusgrow medium(+Amp)

37°C

Transformation

No name

Name	Sample(µL)	Competent Cells(µL)	Total(µL)	Plate
9/5 Pconst+spinach-DT	2	20	22	--
9/5 Plac+spinach-DT	2	20	22	--
9/5 Pconst+pT181 antisense	2	20	22	--
9/5 Plac+pT181 antisense	2	20	22	--
9/5 Ptet+pT181 antisense	2	20	22	--
9/5 Plux+pT181 attenuator	2	20	22	--
9/5 Ptet+pT181 attenuator	2	20	22	--
9/5 Ptet+pT181 attenuator	2	20	22	--
9/5 Pconst+RBS-tetR+DT	2	20	22	--
9/16(2012) T7-His-FT	2	20	22	--
9/16(2012) pBr322	2	20	22	--

Restriction Enzyme Digestion

No name

	9/5 Pbad/araC	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	4µL	0µL	1µL	0µL	1µL	3µL	3µL	18µL	30µL

	9/5 RBS-luxI-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	4µL	0µL	0µL	1µL	1µL	3µL	3µL	18µL	30µL

	8/28 Plux	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	12µL	0µL	1µL	0µL	1µL	3µL	3µL	10µL	30µL
NC	0.6µL	0µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/5 Pconst(J23100)	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	5.7µL	0µL	1µL	0µL	1µL	3µL	3µL	16.3µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/5 Plac	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	1µL	0µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/31 Plux-RBS-GFP-DT-1	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	9.4µL	1µL	1µL	0µL	0µL	3µL	3µL	12.6µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 Pconst-RBS-luxR-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	4.5µL	1µL	0µL	1µL	0µL	3µL	3µL	13.5µL	30µL
NC	0.2µL	0µL	0µL	0µL	0µL	1µL	1µL	7.8µL	10µL

DNA	concentration[µg/mL]	260/280	260/230
9/5 RBS-luxI-DT	7.6	1.56	-1.42
9/5 Pbad/araC	7.5	1.54	3.84

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	aptamer 12_1R (pSB1C3)-1	--	--
3	aptamer 12_1R (pSB1C3)-2	--	--
4	pT181 antisense(pSB1C3)-1	--	--
5	pT181 antisense(pSB1C3)-2	--	--
6	pT181 antisense(pSB1C3)-3	--	--
7	pT181 antisense(pSB1C3)-4	--	--
8	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	aptamer 12_1R-DT-1	--	--
3	aptamer 12_1R-DT-2	--	--
4	aptamer 12_1R-DT-3	--	--
5	aptamer 12_1R-DT-4	--	--
6	pT181 attenuator(pSB1C3)-1	--	--
7	pT181 attenuator(pSB1C3)-2	--	--
8	RBS-lysis1-DT-1	--	--
9	RBS-lysis1-DT-2	--	--
10	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	RBS-lysis2-DT-1	--	--
3	RBS-lysis2-DT-2	--	--
4	RBS-lysis2-DT-3	--	--
5	RBS-lysis2-DT-4	--	--
6	RBS-lysis3-DT-1	--	--
7	RBS-lysis3-DT-2	--	--
8	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	Pconst	SpeI	PstI
3	Pconst NC	--	--
4	Plac	SpeI	PstI
5	Plac NC	--	--
6	Plux	SpeI	PstI
7	Plux NC	--	--
8	Pconst-RBS-luxR-DT	EcoRI	XbaI
9	Pconst-RBS-luxR-DT NC	--	--
10	Plux-RBS-GFP-DT	EcoRI	SpeI
11	Plux-RBS-GFP-DT NC	--	--
12	1kbp ladder	--	--

5x M9 Medium (+EDTA)

Hirano

volume	10ml
Na2HPO4	60mg
KH2PO4	30mg
NaCl	5mg
NH4Cl	10mg
Fe(III)-EDTA	1263.27mg
MilliQ	up to 10 mL

autoclave at 121 °C for 20 min

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder
2	Pconst	SpeI & PstI
3	Pconst	SpeI & PstI

Name	concentration[µg/mL]	260/280	260/230
Pconst	5.3	1.98	0.06

Lane	DNA	Enzyme
1	100bp ladder
2	Plac	SpeI & PstI
3	Plac	SpeI & PstI
5	Plux	SpeI & PstI
6	Plux	SpeI & PstI

Name	concentration[µg/mL]	260/280	260/230
Plac (SpeI & PstI)	5.2	1.82	0.36
Plux(SpeI & PstI)	8.0	1.87	0.25

Lane	DNA	Enzyme
1	1kbp ladder
2	Pconst-RBS-luxR-DT	EcoRI & XbaI
3	Pconst-RBS-luxR-DT	EcoRI & XbaI
5	Plux-RBS-GFP-DT	EcoRI & SpeI
6	Plux-RBS-GFP-DT	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
Pconst-RBS-luxR-DT(EcoRI & XbaI)	30.6	1.84	1.16
Plux-RBS-GFP-DT (EcoRI & SpeI)	21.8	1.87	0.98

DNA Purification

No name

DNA	concentration[µg/mL]	260/280	260/230
9/5 RBS-luxI-DT	7.6	1.56	-1.42
9/5 Pbad/araC	7.5	1.54	3.84

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
8/9 J23100-5	243.0	1.90	1.81

Colony PCR

No name

Sample	base pair
9/4 RBS-lysis3-DT-3	1210
9/4 RBS-lysis3-DT-4	1210
9/4 RBS-lysis3-DT-5	1210
9/4 RBS-lysis1-DT-3	613
9/4 Ptrc KaiC-1	--
9/4 pSB4K5	1370

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min 25s	30cycles

Liquid Culture

Hirano

Sample	medium
9/4 aptamer 12_1R-DT-1	Plusgrow medium (+CP)
9/4 pT181 attenuator(pSB1C3)-1	Plusgrow medium (+CP)
9/4 RBS-lysis2-DT-1	Plusgrow medium (+CP)
9/4 Ptrc-KaiC -1	Plusgrow medium (+Amp)
9/4 pSB4K5 -1	Plusgrow medium (+Kan)

Sep 6

Electrophoresis

Hirano

Lane	Sample
1	100bp ladder
2	9/4 RBS-lysis3-DT -3
3	9/4 RBS-lysis3-DT -4
4	9/4 RBS-lysis3-DT -5
5	9/4 RBS-lysis1-DT -3
6	9/4 Ptrc-KaiC -1
7	9/4 pSB4K5 -1
8	100bp ladder

Colony PCR

No name

Sample	base pair
Pconst-spinach-DT-(1~4)	563
Plac-spinach-DT-(1~4)	659
Pcon-pT181 antisense-(1~4)	468
Ptet-pT181 antisense-(1~4)	604
Pconst-pT181 attenuator-(1~4)	568
Pconst+RBS-tetR-DT-(1~4)	1121
Ptet+RBS-lacZα-DT-(1~4)	1162

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	40s	30cycles

Liquid Culture

No name

Sample	medium
9/4 RBS-Lysis3+DT3	Plusgrow medium(+CP)
9/4 RBS-Lysis1+DT3	Plusgrow medium(+CP)
9/4 pSB4K5	Plusgrow medium(+Kan)

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	Pconst-spinach-DT -(1)
3	Pconst-spinach-DT -(2)
4	Pconst-spinach-DT -(3)
5	Pconst-spinach-DT -(4)
6	Plac-spinach-DT -(1)
7	Plac-spinach-DT -(2)
8	Plac-spinach-DT -(3)
9	Plac-spinach-DT -(4)
10	Pcon-pT181 antisense -(1)
11	Pcon-pT181 antisense -(2)
12	Pcon-pT181 antisense -(3)
13	Pcon-pT181 antisense -(4)

Lane	Sample
1	100bp ladder
2	Ptet-pT181 antisense -(1)
3	Ptet-pT181 antisense -(2)
4	Ptet-pT181 antisense -(3)
5	Ptet-pT181 antisense -(4)
6	Plac-pT181 attenuator -(1)
7	Plac-pT181 attenuator -(2)
8	Plac-pT181 attenuator -(3)
9	Plac-pT181 attenuator -(4)

Miniprep

Honda and Kojima

DNA	concentration [µg/mL]	260/280	260/230
pT181 antisense-(1)	238.7	1.68	1.44
pT181 antisense-(2)	248.8	1.83	1.77
pT181 antisense-(3)	223.2	1.78	1.61
pT181 antisense-(4)	220.8	1.89	1.92
tetR aptamer-(1)	252.5	1.89	1.90
tetR aptamer-(2)	211.2	1.90	1.99
DT181 attenuator	102.2	1.98	2.17
121R aptamer-DT	251.7	1.92	2.01
pSB4K5	212.9	1.80	1.90
RBS-lacZα-DT	185.7	1.78	1.79
RBS-lysis2-DT-(1)	84.8	1.85	2.24

Restriction Enzyme Digestion

Tatsui

	9/6 RBS-lysis2-DT (78.3 µg/mL)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	22 µL	1 µL	1 µL	3 µL	3 µL	0 µL	30 µL
NC	1.3 µL	0 µL	0 µL	1 µL	1 µL	6.7 µL	10 µL

	aptamer-DT (251.7 µg/mL)	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	0.4 µL	1 µL	1 µL	3 µL	3 µL	14.1 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	Pλ-luxI (86.4 µg/mL)	EcoRI	SpeI	BSA	Buffer	MilliQ	total
2 cuts	23.1 µL	1 µL	1 µL	3 µL	3 µL	1.9 µL	30 µL
NC	1.2 µL	0 µL	0 µL	1 µL	1 µL	6.8 µL	10 µL

at 37 °C, for 1h

Electrophoresis

Hirano

Lane	Sample
1	100bp ladder
2	Pconst+RBS-tetR-DT -(1)
3	Pconst+RBS-tetR-DT -(2)
4	Pconst+RBS-tetR-DT -(3)
5	Pconst+RBS-tetR-DT -(4)
6	Ptet+RBS-lacZα-DT -(1)
7	Ptet+RBS-lacZα-DT -(2)
8	100bp ladder
9	Ptet+RBS-lacZα-DT -(3)
10	Ptet+RBS-lacZα-DT -(4)
11	100bp ladder

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	RBS-lysis2-DT	XbaI & PstI
3	RBS-lysis2-DT	XbaI & PstI
4	--	--
5	aptamer 12_1R-DT	XbaI & PstI
6	aptamer 12_1R-DT	XbaI & PstI

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pλ-luxI	EcoRI & SpeI
3	Pλ-luxI	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis2-DT (XbaI & PstI)	7.1	1.88	0.07
aptamer 12_1R-DT (XbaI & PstI)	15.3	1.93	0.70
Pλ-luxI (EcoRI & SpeI)	4.5	1.79	0.31

Liquid Culture

No name

Sample	medium
9/5 Pconst-spinach-DT-(1)	Plusgrow medium(+Amp&CP)
9/5 Ptet-pT181 antisense-(1)	Plusgrow medium(+CP)
9/5 Pconst+RBS-tetR-DT-(1)	Plusgrow medium(+Amp&CP)
9/5 Ptet+RBS-lacZα-DT-(1)	Plusgrow medium(+CP)
9/5 Pcon-pT181 attenuator-(1)	Plusgrow medium(+CP)
9/4 pT181 antisense-(1)	Plusgrow medium(+CP)
9/4 aptamer 12_1R-(1)	Plusgrow medium(+CP)

Colony PCR

No name

Sample	base pair
9/5 Plac-spinach-DT-(5~12)	563
9/5 Pcon-antisense-(5~12)	468
9/5 Plac-attenuator-(5~12)	664
9/5 pT181 antisense+Plac-(1)	-

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	42s	30cycles

Sep 7

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
RBS-lysis1-DT	195.3
RBS-lysis3-DT	255.5	1.91	2.04
Ptet-pT18	183.6	1.85	1.48
Pcon-RBS-tetR-DT	134.3	1.94	2.20
Ptet-RBS-lacZα-DT	67.7	1.92	1.79
Pcon-pT181 attenuator	232.3	1.95	1.90
pSB4K5	237.0	1.61	1.50
pT181 antisense(pSB1C3)	256.4	1.63	1.78
apt12_1R(pSB1C3)	230.0	1.98	2.28

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	Plac-Spinach-DT -5
3	Plac-Spinach-DT -6
4	Plac-Spinach-DT -7
5	Plac-Spinach-DT -8
6	Plac-Spinach-DT -9
7	Plac-Spinach-DT -10
8	100bp ladder
1	100bp ladder
2	Plac-Spinach-DT -11
3	Plac-Spinach-DT -12
4	Pcon-antisense -5
5	Pcon-antisense -6
6	Pcon-antisense -7
7	Pcon-antisense -8
8	100bp ladder

Lane	Sample
1	100bp ladder
2	Pcon-antisense -9
3	Pcon-antisense -10
4	Pcon-antisense -11
5	Pcon-antisense -12
6	Plac-attenuator -5
7	Plac-attenuator -6
8	100bp ladder
1	100bp ladder
2	Plac-attenuator -7
3	Plac-attenuator -8
4	Plac-attenuator -9
5	Plac-attenuator -10
6	Plac-attenuator -11
7	Plac-attenuator -12
8	100bp ladder

Lane	Sample
1	100bp ladder
2	Plac-pT181 antisense

Colony PCR

No name

Sample
Plac-spinach-DT -13~20
Plac-pT181 attenuator -13~20

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68°C	--
5 min	30 s	30 s	42 sec	30 cycles

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/6 Plux (SpeI&PstI)	2 µL	9/7 RBS-lysis2-DT (EcoRI&XbaI)	µL	3.5 µL
experiment	9/6 Plac (SpeI&PstI)	µL	9/7 RBS-lysis2-DT (EcoRI&XbaI)	µL	3.5 µL
experiment	9/6 Pcon-RBS-luxR-DT (EcoRI&XbaI)	1.9 µL	9/6 Plux-RBS-GFP-DT(SpeI&PstI)	2.2 µL	3.5 µL
experiment	9/6 Pbed/araC (SpeI&PstI)	1.6 µL	9/5 RBS-luxI-DT (EcoRI&XbaI)	16 µL	3.5 µL
experiment	9/6 Plac (SpeI&PstI)	1.5 µL	9/5 pT181 antisense (EcoRI&XbaI)	2.0 µL	3.5 µL
experiment	9/6 Pcon (SpeI&PstI)	3.0 µL	9/5 RBS-luxI-DT (EcoRI&XbaI)	10 µL	3.5 µL

Electrophoresis

Nakamoto

Lane	Sample
1	100bp ladder
2	Plac-Spinach-DT -13
3	Plac-Spinach-DT -14
4	Plac-Spinach-DT -15
5	Plac-Spinach-DT -16
6	Plac-Spinach-DT -17
7	Plac-Spinach-DT -18
8	Plac-Spinach-DT -19
9	Plac-Spinach-DT -20
10	Plac-pT181 attenuator -13
11	Plac-pT181 attenuator -14
12	100bp ladder

Lane	Sample
1	100bp ladder
2	Plac-pT181 attenuator -15
3	Plac-pT181 attenuator -16
4	Plac-pT181 attenuator -17
5	Plac-pT181 attenuator -18
6	Plac-pT181 attenuator -19
7	Plac-pT181 attenuator -20
8	100bp ladder

Transformation

Tatsui

Name	Sample	Competent Cells	Total
Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT	2µL	20µL	22µL
Pbed/araC+RBS-luxI-DT	2µL	20µL	22µL
Plac+pT181 antisense	2µL	20µL	22µL
Pcon+RBS-luxI-DT	2µL	20µL	22µL

Restriction Enzyme Digestion

No Name

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/7 RBS-lysis1-DT(XbaI & PstI)	10 µL	1 µL	1 µL	3 µL	3 µL	12 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/7 RBS-lysis3-DT (XbaI & PstI)	7.8 µL	1 µL	1 µL	3 µL	3 µL	14.2 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	DNA	SpeI	PstI	Buffer	BSA	MilliQ	total
9/5 Plac (SpeI & PstI)	6.0 µL	1 µL	1 µL	3 µL	3 µL	16.0 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	EcorI	SpeI	Buffer	BSA	MilliQ	total
9/7 pSB4K5 (EcoRI & SpeI)	8.4 µL	1 µL	1 µL	3 µL	3 µL	16.0 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	restricion enzyme	restriction enzyme	Buffer	BSA	MilliQ	total
Pcon-pT181 attenuator (SpeI & PstI)	8.6 µL	1 µL	1 µL	3 µL	3 µL	13.4 µL	30 µL
Pcon-pT181 attenuator (EcorI & SpeI)	8.6 µL	1 µL	1 µL	3 µL	3 µL	13.4 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/5 Spinach-DT (XbaI & PstI)	5.4 µL	1 µL	1 µL	3 µL	3 µL	16.6 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	SpeI	PstI	Buffer	BSA	MilliQ	total
Ptet-anticodone (SpeI & PstI)	11 µL	1 µL	1 µL	3 µL	3 µL	11 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/6 RBS-lacZα-DT (XbaI & PstI)	11 µL	1 µL	1 µL	3 µL	3 µL	11 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	EcoRI	SpeI	Buffer	BSA	MilliQ	total
pSB1C3 (1) (EcoRI & SpeI)	6.6 µL	1 µL	1 µL	3 µL	3 µL	15.4 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
pSB1C3 (3) (XbaI & PstI)	6.6 µL	1 µL	1 µL	3 µL	3 µL	15.4 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	RBS-lysis1-DT (9/7)	XbaI	PstI
3	RBS-lysis1-DT (9/7)	--	--
4	RBS-lysis3-DT (9/7)	XbaI	PstI
5	RBS-lysis3-DT (9/7)	--	--
6	Pcon-pT181 attenuator	EcoRI	SpeI
7	Pcon-pT181 attenuator	--	--
8	9/5 Spinach-DT	XbaI	PstI
9	9/5 Spinach-DT	--	--
10	100bp ladder	--	--
11	100bp ladder	--	--
12	RBS-lacZα-DT	XbaI	PstI
13	RBS-lacZα-DT	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Ptet-antisense	SpeI	PstI
3	Plac (NC)	--	--
4	pSB4K5	EcoRI	SpeI
5	pSB4K5	--	--
6	Pcon-pT181 attenuator	SpeI	PstI
7	1kbp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Plac	SpeI	PstI
3	Ptet-antisense	--	--
4	pSB1C3	EcoRI	SpeI
5	pSB1C3	--	--
6	pSB1C3	XbaI	PstI
7	pSB1C3	--	--
8	1kbp ladder	--	--

Liquid Culture

No name

Sample	medium
entA- strain	SOB medium (+Kan)
9/15 Pcon-pT181 antisense	Plusgrow (+Amp)

Sep 8

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder
2	RBS-lysis1-DT	XbaI & PstI
3	RBS-lysis1-DT	XbaI & PstI
5	RBS-lysis3-DT	XbaI & PstI
6	RBS-lysis3-DT	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis1-DT (XbaI & PstI)	5.3	1.98	0.06
RBS-lysis3-DT (XbaI & PstI)	5.3	1.98	0.06

Lane	DNA	Enzyme
1	100bp ladder
3	Pcon-pT181 attenuator	EcoRI & SpeI
4	Pcon-pT181 attenuator	EcoRI & SpeI
6	Spinach-DT	XbaI & PstI
7	Spinach-DT	XbaI & PstI
9	RBS-lacZα-DT	XbaI & PstI
10	RBS-lacZα-DT	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator (EcoRI & SpeI)	5.2	1.82	0.36
Spinach-DT (XbaI & PstI)	8.0	1.87	0.25
RBS-lacZα-DT (XbaI & PstI)	10.3	1.71	0.54

Lane	DNA	Enzyme
1	1kbp ladder
2	Ptet-pT181 antisense	EcoRI & PstI
3	Ptet-pT181 antisense	EcoRI & PstI
5	pSB4K5	EcoRI & SpeI
6	pSB4K5	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
Ptet-pT181 antisense(EcoRI & PstI)	30.6	1.84	1.16
pSB4K5 (EcoRI & SpeI)	21.8	1.87	0.98

Lane	DNA	Enzyme
1	1kbp ladder
2	Pcon-pT181 attenuator	SpeI & PstI
3	Pcon-pT181 attenuator	SpeI & PstI
5	Plac	SpeI & PstI
6	Plac	SpeI & PstI
8	pSB1C3	EcoRI & SpeI
9	pSB1C3	EcoRI & SpeI
11	pSB1C3	XbaI & PstI
12	pSB1C3	XbaI & PstI

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator (SpeI & PstI)	14.5	1.91	1.09
Plac (SpeI & PstI)	11.6	1.74	1.19
pSB1C3 (EcoRI & SpeI)	5.7	1.74	0.34
pSB1C3 (XbaI & PstI)	5.6	2.00	0.37

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pcon-pT181 anti	243.0	1.90	1.81
Pcon-Spinach-DT	253.7	1.57	1.51

Ligation

Kojima

state	Vector(µL)		Inserter(µL)		Ligation High ver.2
experiment	9/8 Plac SpeI+PstI	4.3	9/3 pT181 attenuator	3.1	3.5
experiment	9/8 Plac SpeI+PstI	4.3	9/7 aptamer12-1R-DT XbaI+PstI	2.1	3.2
experiment	9/8 Plac SpeI+PstI	4.3	9/8 spinach-DT XbaI+PstI	4.6	3.5
experiment	9/6 Plac SpeI+PstI	2	9/7 RBS-lysis2-DT XbaI+PstI	10	3.5
experiment	9/8 Plac SpeI+PstI	4.3	9/7 RBS-lysis2-DT XbaI+PstI	10	3.5
experiment	9/8 Ptet-pT181 anti	1.6	9/8 spinach-DT XbaI+PstI	4.3	3.0
experiment	9/6 Pcon SpeI+PstI	1.4	9/8 RBS-laczα-DT XbaI+PstI	4.8	3.1
experiment	9/8 Pcon-pT181 attenuator SpeI+PstI	3.4	9/7 aptamer12-1R-DT XbaI+PstI	1.9	2.7
experiment	9/6 Pcon SpeI+PstI	1.4	9/7 aptamer12-1R-DT XbaI+PstI	2.1	1.3
experiment	9/8 DT	3.0	9/8 Pcon-pT181 attenuator EcoRI+SpeI	7.9	3.5
experiment	9/6 Plux SpeI+PstI	2	9/7 RBS-lysis1-DT XbaI+PstI	7.2	3.5
experiment	9/8 Plac SpeI+PstI	4.3	9/7 RBS-lysis1-DT XbaI+PstI	7.2	3.5
experiment	9/4 Plac SpeI+PstI	1.7	9/8 RBS-laczα-DT XbaI+PstI	4.8	3.3

Restriction Enzyme Digestion

Kojima

	9/8 Pcon-pT181 anti	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	8.2µL	0µL	1µL	0µL	1µL	3µL	3µL	13.8µL	30µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/8 Pcon-spinach-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	7.9µL	1µL	1µL	0µL	0µL	3µL	3µL	14.1µL	30µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/21 F1 attenuator	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.5µL	0µL	0µL	1µL	1µL	3µL	3µL	15.5µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F3m2 attenuator	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	0µL	1µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F1 anti	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.7µL	0µL	0µL	1µL	1µL	3µL	3µL	15.3µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 F6 anti	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.0µL	0µL	0µL	1µL	1µL	3µL	3µL	16µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 tetR aptamer 12_1M	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	5.3µL	1µL	1µL	0µL	0µL	3µL	3µL	16.7µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	8/21 tetR aptamer 12_P	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	13.3µL	1µL	1µL	0µL	0µL	3µL	3µL	8.7µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/4 spinach-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	5.4µL	0µL	0µL	1µL	1µL	3µL	3µL	16.6µL	30µL
NC	0.3µL	0µL	0µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/7 RBS-lysis1-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	10µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/6 RBS-lysis2-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	13µL	0µL	0µL	1µL	1µL	3µL	3µL	9µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/7 RBS-lysis3-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	10µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 J23100 RBS-lacZα-DT-(1)	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	7µL	0.5µL	0.5µL	0µL	0µL	3µL	3µL	16µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	8/20 RBS-lacZα-DT	EcoRI	SpeI	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	10µL	0.5µL	0.5µL	0µL	0µL	3µL	3µL	13µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

Transformation

Nakamoto & Ashida

Name	Sample(µL)	Competent Cells(µL)	Total(µL)	Plate
9/8 Pcont+aptamer 12-1R-DT	1	10	11	Amp
9/8 Pcon+RBS-lacZα-DT	1	10	11	Amp
9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT	1	10	11	Amp
9/8 Plac+RBS-lysis1-DT	2	20	22	CP
9/8 Plac+RBS-lysis2-DT	2	20	22	CP
9/8 Plac+aptamer 12-1R-DT	2	20	22	CP
9/8 Plac+spinach	2	20	22	CP
9/8 Plac+pT181 attenuator	2	20	22	CP
9/8 Plux+RBS-lysis1-DT	1	10	11	CP
9/8 Plux+RBS-lysis2-DT	1	10	11	CP
9/8 Ptet+RBS-lacZα-DT	1	10	11	CP
9/8 Ptet-pT181 anti+spinach-DT	1	10	11	CP
9/8 Pcon-pT181 attenuator+DT	1	10	11	CP

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder
2	Pcon-spinach-DT	EcoRI	SpeI
3	Pcon-spinach-DT
4	Fusion1 attenuator	XbaI	PstI
5	Fusion1 attenuator
6	Fusion3m2 attenuator	XbaI	PstI
7	Fusion3m2 attenuator
8	Fusion1 anti	XbaI	PstI
9	Fusion1 anti
10	Fusion6 anti	XbaI	PstI
11	Fusion6 anti
12	aptamer 12-1M	XbaI	PstI
13	aptamer 12-1M
14	aptamer 12-P	XbaI	PstI
15	aptamer 12-P
16	spinach-DT	XbaI	PstI

Lane	Sample	Enzyme1	Enzyme2
1	spinach-DT
2	J23100-RBS-lacZα-DT	EcoRI	SpeI
3	J23100-RBS-lacZα-DT
4	100bp ladder
5	RBS-lacZα-DT	EcoRI	SpeI
6	RBS-lacZα-DT
7	RBS-lysis1-DT	XbaI	PstI
8	RBS-lysis1-DT

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder
2	RBS-lysis2-DT	XbaI	PstI
3	RBS-lysis2-DT
4	RBS-lysis3-DT	XbaI	PstI
5	RBS-lysis3-DT
6	Pcon pT181 anti	SpeI	PstI
7	Pcon pT181 anti

Gel Extraction

Ashida and Nakamoto

Lane	DNA	Enzyme
1	100bp ladder
2	Pcon-spinach-DT	EcoRI+SpeI
3	Pcon-Spinach-DT	EcoRI+SpeI
5	Fusion 1 attenuator	XbaI+PstI
6	Fusion 1 attenuator	XbaI+PstI
8	Fusion3m2 attenuator	XbaI+PstI
9	Fusion3m2 attenuator	XbaI+PstI
11	Fusion 1 antisense	XbaI+PstI
12	Fusion 1 antisense	XbaI+PstI

Name	concentration[µg/mL]	260/280	260/230
Pcon-spinach-DT(EcoRI+SpeI)	10.5	1.86	0.39
Fusion 1 attenuator(XbaI+PstI)	7.0	1.71	0.27
Fusion3m2 attenuator(XbaI+PstI)	8.6	1.75	0.47
Fusion 1 antisense(XbaI+PstI)	7.6	1.87	0.43

Lane	DNA	Enzyme
1	100bp ladder
2	Fusion6 antisense	XbaI+PstI
3	Fusion6 antisense	XbaI+PstI
5	aptamer12_1M	XbaI+PstI
6	aptamer12_1M	XbaI+PstI

Name	quantity	concentration[µg/mL]	260/280	260/230
Fusion6 antisense(XbaI+PstI)		5.0	1.76	0.20
aptamer12_1M(XbaI+PstI)		3.3	2.31	0.04

Lane	DNA	Enzyme
1	100bp ladder
2	aptamer12_P	XbaI+PstI
3	aptamer12_P	XbaI+PstI
5	spinach-DT	EcoRI+SpeI
6	spinach-DT	EcoRI+SpeI

Name	quantity	concentration[µg/mL]	260/280	260/230
aptamer12_P(XbaI+PstI)		3.4	1.72	0.26
spinach-DT(EcoRI+SpeI)		10.1	1.57	0.47

Lane	DNA	Enzyme
1	100bp ladder
2	J23100-RBS-laczα-DT	EcoRI+SpeI
3	J23100-RBS-laczα-DT	EcoRI+SpeI
5	RBS-laczα-DT	EcoRI+SpeI
6	RBS-laczα-DT	EcoRI+SpeI
8	RBS-lysis1-DT	XbaI+PstI
9	RBS-lysis1-DT	XbaI+PstI
11	RBS-lysis2-DT	XbaI+PstI
12	RBS-lysis2-DT	XbaI+PstI

Name	concentration[µg/mL]	260/280	260/230
Pcon-RBS-laczα-DT(EcoRI+SpeI)	5.6	2.03	0.25
RBS-laczα-DT(EcoRI+SpeI)	4.4	2.04	0.33
RBS-lysis1-DT(XbaI+PstI)	5.9	2.22	0.53
RBS-lysis2-DT(XbaI+PstI)	5.1	1.67	0.48

Lane	DNA	Enzyme
1	100bp ladder
2	RBS-lysis3-DT	XbaI+PstI
3	RBS-lysis3-DT	XbaI+PstI
5	Pcon-pT181 antisense	SpeI+PstI
6	Pcon-pT181 antisense	SpeI+PstI

Name	quantity	concentration[µg/mL]	260/280	260/230
RBS-lysis3-DT(XbaI+PstI)		23.4	1.72	0.88
Pcon-pT181 antisense(SpeI+PstI)		31.3	1.86	0.92

Ligation

No name

state	Vector		Inserter
experiment	Pcon-spinach-DT(EcoRI&SpeI)	10.5µg/mL	pSB6A1(EcoRI&SpeI)	8.9µg/mL
experiment	Pcon-spinach-DT(EcoRI&SpeI)	10.5µg/mL	pSB4K5(EcoRI&SpeI)	21.8µg/mL
experiment	Pcon-pT181 anti(SpeI+PstI)	31.3µg/mL	spinach-DT(XbaI+PstI)	10.1µg/mL
experiment	Plac(SpeI+PstI)	11.6µg/mL	RBS-lysis3-DT	23.4µg/mL
experiment	Plux(SpeI+PstI)	25µg/mL	RBS-lysis3-DT	23.4µg/mL
experiment	Ptet-pT181 anti(SpeI+PstI)	30.6µg/mL	spinach-DT(XbaI+PstI)	10.1µg/mL
experiment	Pcon-pT181 attenuator(SpeI+PstI)	14.5µg/mL	RBS-laczα-DT(XbaI+PstI)	4.4µg/mL

PCR

No name

KaiA(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(661-RBS-KaiA-fwd)	Primer(kaiA-662-rev-ver2)	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

KaiB(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(662-RBS-KaiB-fwd-ver2)	Primer(kaiB-663-rev-ver2)	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

DT(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(664-DT-fwd-ver2)	Primer(DT-suffix-660-rev-ver2)	MilliQ	total
0.1	0.5	2.5	2.5	1.5	0.75	0.75	16.4	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

P-KaiB(PCR prpduct)	KOD plus	10x buffer	dNTP	MgSO4	Primer(prefix-PkaiBC-fwd-ver2)	Primer(PkaiBC-suffix--662-rev-ver2)	MilliQ	total
0.7	0.5	2.5	2.5	1.5	0.75	0.75	15.8	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	28s	30cycles

Plac(1)(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(GG0-pSBIC3-fwd-ver2)	Primer(Plac-GG1-rev-ver2)	MilliQ	total
0.13	0.5	2.5	2.5	1.5	0.75	0.75	16.37	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	60s	30cycles

KaiC(plasmid)	KOD plus	10x buffer	dNTP	MgSO4	Primer(GG3-RBS-KaiC-fwd-ver2)	Primer(KaiC-GG4-rev-ver2)	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	60s	30cycles

Gel Extraction

Yoshida

Lane	DNA
1	100bp ladder
2	KaiA
3	KaiA
4	KaiB
5	KaiB
6	DT
7	DT
8	P-KaiBC
9	P-KaiBC
10	nega
11	100bp ladder

Lane	DNA
1	Plac
2	Plac
3	KaiC
4	KaiC
5	nega
6	1kb ladder

Sep 9

PCR

Nakagawa

KaiA(plasmid)	10x buffer	dNTPs	MgSO4	Primer(661-RBS-KaiA-fwd)	Primer(kaiA-662-rev-ver2)	Kod-plus ver2	MilliQ	total
1.0	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

KaiB(plasmid)	10x buffer	dNTPs	MgSO4	Primer(661-RBS-KaiA-fwd-ver2)	Primer(kaiB-663-rev-ver2)	Kod-plus ver2	MilliQ	total
1.0	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

P-KaiBC(PCR products)	10x buffer	dNTPs	MgSO4	Primer(prefix-P-kaiBC-fwd-ver2)	Primer(P-KaiBC-suffix-rev-verw)	Kod-plus ver2	MilliQ	total
0.7	2.5	2.5	1.5	0.75	0.75	0.5	15.8	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

DT(plasmid)	10x buffer	dNTPs	MgSO4	Primer(664-DT-fwd-ver2)	Primer(DT-suffix-660-rev-ver2)	Kod-plus ver2	MilliQ	total
1.0	2.5	2.5	1.5	0.75	0.75	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58.5°C	68°C	--
2min	10s	30s	30s	30cycles

Gel Extraction

Yoshida

Lane	DNA	Enzyme
1	100bp ladder	--
2	KaiA	--
3	KaiA	--
4	KaiB	--
5	KaiB	--
6	P-KaiBC	--
7	P-KaiBC	--
8	DT	--
9	DT	--
10	NC	--
11	100bp ladder	--

Name	concentration[µg/mL]	260/280	260/230
KaiC	28.5	1.26	11.92
Plac	53.9	1.82	1.61
KaiB	59.6	1.61	2.20
KaiB	76.5	1.86	1.44
DT	98.7	1.47	1.43

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/8 pSB1C3	83.2	1.45	0.76
9/8 RBS-lysis2-DT	72.8	1.67	0.97
9/8 spinach-DT	152.6	1.88	1.97
Nuclease-Free water	-0.4	-14.30	0.25

Restriction Enzyme Digestion

No name

	9/9 pSB1C3	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	24µL	1µL	1µL	3µL	3µL	22µL	30µL*
NC	1.2µL	0µL	0µL	1µL	1µL	6.8 µL	10µL

	9/9 pSB1C3	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	27.4µL	1µL	1µL	3µL	3µL	22µL	30µL*
NC	1.3µL	0µL	0µL	1µL	1µL	6.7µL	10µL

	9/9 spinach-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	10.8µL	1µL	1µL	3µL	3µL	22µL	30µL*
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

"*"=be evaporated

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
Pcon+RBS-luxI-DT	2µL	20µL	22µL	Amp
Pcon-RBS-luxR-DT+Plux-GFP	2µL	20µL	22µL
Ptet-PT181 antisense+spinach-DT-Pbad/araC+RBS-lux-DT	2µL	20µL	22µL

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/8 F1-attenuator(XbaI&PstI)	5.3µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 F3m2-attenuator(XbaI&PstI)	4.5µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 F1-antisense(XbaI&PstI)	1.7µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 F6-antisense(XbaI&PstI)	2.9µg	9/8 pSB1C3(XbaI&PstI)	8.9µg	3.5µg
experiment	9/8 aptamer12-P(EcoRI&SpeI)	3.1µg	9/8 pSB1C3(EcoRI&SpeI)	8.8µg	3.5µg
experiment	9/8 aptamer12-1M(EcoRI&SpeI)	3.1µg	9/8 pSB1C(EcoRI&SpeI)3	8.8µg	3.5µg

M9 medium

Shimizu & Hirano

EDTA consenlation	Na2HPO4	KH2PO4	NaCl	NH4Cl	Fe(Ⅲ)-EDTA	distilled water	total
1mMol	300mg	150mg	25mg	50mg	21mg	up to 10mL	10mL
10mMol	300mg	150mg	25mg	50mg	210.5mg	up to 10mL	10mL
50mMol	300mg	150mg	25mg	50mg	1052.7mg	up to 10mL	10mL

Colony PCR

No name

Sample	base pair
9/8 Plac-spinach-DT-(1)	659
9/8 Plac-spinach-DT-(2)	659
9/8 Pcon-pt181attenuator-DT-(1)	584

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	40s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2	Spinach-DT	XbaI+PstI
3	Spinach-DT	XbaI+PstI
5	RBS-lacZα-DT	XbaI+PstI
6	RBS-lacZα-DT	XbaI+PstI

Lane	DNA	Enzyme
1	100bp ladder	--
3	pSB1C3	XbaI+PstI
4	pSB1C3	XbaI+PstI

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	pSB1C3(XbaI+PstI)	xx µL	9/8 F1-attenuator(XbaI+PstI)	xx µL	xx µL
experiment	pSB1C3(XbaI+PstI)	xx µL	9/8 F3m2-attenuator(XbaI+PstI)	xx µL	xx µL

PCR

Tatsui and Yoshida

KaiC	10x buffer KOD plus ver2	2M dNTP	MgSO4	Primer Mix(F&R)	Kod-plus ver2	MilliQ	total
0.5	2.5	2.5	1.5	1.5	0.5	16.0	25.0
1.0	2.5	2.5	1.5	1.5	0.5	15.5	25.0
2.0	2.5	2.5	1.5	1.5	0.5	14.5	25.0
4.0	2.5	2.5	1.5	1.5	0.5	12.5	25.0

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	2min	30cycles

Plac(100pg)	10x buffer KOD plus ver2	2M dNTP	MgSO4	Primer Mix(F&R)	Kod-plus ver2	MilliQ	total
0.3	2.5	2.5	1.5	1.5	0.5	16.2	25.0
0.6	2.5	2.5	1.5	1.5	0.5	15.9	25.0
1.2	2.5	2.5	1.5	1.5	0.5	15.3	25.0
2.4	2.5	2.5	1.5	1.5	0.5	14.1	25.0
0.0	2.5	2.5	1.5	1.5	0.5	16.5	25.0

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	2min	30cycles

Liquid Culture

No name

Sample	medium
pSB2C3(master)-2	Plusgrow medium(+CP)

Restriction Enzyme Digestion

No name

	8/17 lysis1-2	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	8.1µL	1µL	1µL	3µL	3µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/17 lysis3-1	XbaI	PstI	BSA	Buffer	MilliQ	total
2 cuts	6.1µL	1µL	1µL	3µL	3µL	15.9µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/15 Plac	XbaI	PstI	BSA	Buffer	MilliQ	total
1 cuts	12.1µL	0µL	1µL	3µL	3µL	10.9µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

PCR

Yoshida

KaiA(KaiABC) 100pg/µL	10x buffer KOD plus ver2	dNTPs	MgSO4	Primer Mix(Fwd&Rev)	KOD plus ver2	MilliQ	total
2.0	2.5	2.5	1.5	1.5	0.5	14.5	25
4.0	2.5	2.5	1.5	1.5	0.5	12.5	25
8.0	2.5	2.5	1.5	1.5	0.5	8.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	--
2min	10s	30s	1min	30cycles

PKaiBC 400pg/µL	10x buffer KOD plus ver2	dNTPs	MgSO4	Primer Mix(Fwd&Rev)	KOD plus ver2	MilliQ	total
0.5	2.5	2.5	1.5	1.5	0.5	16.0	25
1.0	2.5	2.5	1.5	1.5	0.5	15.5	25
2.0	2.5	2.5	1.5	1.5	0.5	14.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	--
2m	10s	30s	1m	30cycles

Liquid Culture

No name

Sample	medium
9/2 Spinach-DT	Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT	Plusgrow medium
9/6 master plate	Plusgrow medium(+Amp)
3 Pcon-PT181 attenuator	Plusgrow medium

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	lysis1	XbaI+PstI
4
5
6

Lane	DNA	Enzyme
1	1kbp ladder	--
3	lysis3	XbaI+PstI
4
5
6

Name	concentration[µg/mL]	260/280	260/230
（´ω｀）
（ΦωΦ^）

Electrophoresis

Yoshida

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	Plac 100pg	--	--
3	Plac 200pg	--	--
4	Plac 400pg	--	--
5	Plac 800pg	--	--
6	NC	--	--
7	1kbp ladder	--	--

Sep 10

Ligation

Nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/6 Pcon-RBS-luxR-DT	1.9µL	9/6Plux-RBS-GFP-DT	22 µL	3.5µL
experiment	9/6 Plux	2µL	9/8 RBS-lysis1-DT	6.6µL	3.5µL
experiment	9/6 Plux	2µL	9/8 RBS-lysis2-DT	6.3µL	3.5µL
experiment	9/8 Ptet-PT181 antisense	1.6µL	9/8 spinach-DT	4.4µL	3.0µL
experiment	9/8 Pcon-PT181 attenuator	3.4µL	9/8 apz 12-1R-PT	2.1µL	2.8µL
experiment	9/8 Pcon	3.4µL	9/8 apz 12-1R-PT	2.3µL	2,9µL
experiment	9/8 DT	3.0µL	9/8 Pcon-PT181 attenuator	7.9µL	3.5µL
experiment	9/10 pSB1C3	1.8µL	9/8 F3m2 attenuator	4.6µL	3.2µL
experiment	9/10 pSB1C3	1.8µL	9/8 F1 attenuator	3.4µL	2.6µL

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
spinach-DT	202.4	1.97	1.93
Plux-RBS-GFP-DT	164.2	2.80	2.06
Pcon-DT attenuator	205.4	1.81	2.39
RSBJC3-2	240.8	1.73	1.43
Plaz-1	240.8	1.73	1.43

Restriction Enzyme Digestion

Nakamoto

	9/10　Plux-RBS-GFP-DT	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	12.1µL	1µL	1µL	3µL	3µL	9.9µL	30µL
NC	0.6µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/10　Spinach-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	9.9µL	1µL	1µL	3µL	3µL	12.1µL	30µL
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/6 apt12-1R -DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	8.0µL	1µL	1µL	3µL	3µL	14.0µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/10　Pcon-pt81 attenuator	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	9.8µL	1µL	1µL	3µL	3µL	12.2µL	30µL
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Plux-RBS-GEP-DT	EcoRI	SpeI
3	Plux-RBS-GEP-DT	--	--
4	Spinach-DT	XbaI	PstI
5	Spinach-DT	--	--
6	aptamer 121R-DT	XbaI	PstI
7	aptamer 121R-DT	--	--
8	Pcon-pt181 attenuator	EcoRI	SpeI
9	Pcon-pt181 attenuator	--	--
10	100bp ladder	--	--

Colony PCR

Tatsui

Sample	base pair
9/9 Pcon-pT181 antisense	--
9/8 J223100(entA)	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min10sec	30

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	Pcon Pt181 antisense1+spinach-DT
3	Pcon Pt181 antisense2+spinach-DT
4	Pcon Pt181 antisense3+spinach-DT
5	Pcon Pt181 antisense4+spinach-DT
6	Pcon Pt181 antisense5+spinach-DT
7	Pcon Pt181 antisense6+spinach-DT
8	Pcon Pt181 antisense7+spinach-DT
9	Pcon Pt181 antisense8+spinach-DT
10	Pcon Pt181 antisense9+spinach-DT
11	Pcon Pt181 antisense10+spinach-DT
12	Pcon Pt181 antisense11+spinach-DT
13	Pcon Pt181 antisense12+spinach-DT
14	100bp ladder

Lane	Sample
1	1kbp ladder
2	Pcon entA1
3	Pcon entA2
4	Pcon entA3
5	1kbp ladder

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kbp ladder	--
2	KaiA(200pg)	--
3	KaiA(200pg)	--
4	--	--
5	KaiA(400pg)	--
6	KaiA(400pg)	--
7	--	--
8	KaiA(800pg	--
9	KaiA(800pg)	--
10	1kbp ladder	--

Lane	DNA	Enzyme
1	1kbp ladder	--
2	PKaiBC(200pg)	--
3	PKaiBC(200pg)	--
4	--	--
5	PKaiBC(400pg)	--
6	PKaiBC(400pg)	--
7	--	--
8	PKaiBC(800pg	--
9	PKaiBC(800pg)	--
10	1kbp ladder	--

Liquid Culture

Hirano

Sample	medium
9/4 J23100①(JM109)	LB(Amp)
9/8 J23100① (entA)	LB(Amp)

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kbp ladder	--
2	KaiC	--
3	KaiC	--
4	KaiC	--
5	--	--
6	1kbp ladder	--
7	1kbp ladder	--
8	KaiC	--
9	KaiC	--
10	KaiC	--
11	--	--
12	1kbp ladder	--

Liquid Culture

Tatsui

Sample	medium
9/10 Pcon-pt181 antisense-Spinach-DT	Plusgrow

Colony PCR

No name

Sample	base pair
9/9 Phad larac-1 RBS-lux1-DT	2331

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30

Sample	base pair
9/9 Pcon-lacZα (psB4KS)	712
9/9 Pcon-Spinach-DT(psB4KS)	605

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	42s	30

Sample	base pair
9/9 Plac+RBS-lysis3}-DT	1273
9/9 Pcon+RBS-lux1-DT	1079

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min12s	30

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	9/9 Pcou-RBS-lacZα-DT(p3B4KS9)-1	--	--
3	9/9 Pcou-RBS-lacZα-DT(p3B4KS9)-2	--	--
4	9/9 Pcou-Spinach-DT(pB4KS)-1	--	--
5	9/9 Pcou-Spinach-DT(pB4KS)-2	--	--
6	100bp ladder	--	--

Transformation

Nakamoto

Name	Plate
F1 attenuator +p3B1C3	CP
F3n2 attenuator +p3B1C3	CP
F1 antisense +p3B1C3	CP
F6 antisense+p3B1C3	CP
apt12-P +p3B1C3	CP
apt12-1M +p3B1C3	CP
Pcon-luxR+Plux-GFP	Amp
Plux+RBS-lysis1-DT	CP
Plux+RBS-lysis2-DT	CP
Ptet-pT181antisense+Spinach-DT	CP
Pcon-pT181 attenuator+apt121R-DT	Amp
Pcon-1 apt12-1R-DT	Amp
DT+Pcon-pT181 attenuator	CP

Electrophoresis

Okazaki

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Spinach-DT	XbaI	PstI
3	Spinach-DT	XbaI	PstI
4	Spinach-DT	XbaI	PstI
5	Spinach-DT	XbaI	PstI
6	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-pT181 attenuator	EcoRI	SpeI
3	Pcon-pT181 attenuator	EcoRI	SpeI
4	Pcon-pT181 attenuator	EcoRI	Spei
5	Pcon-pT181 attenuator	EcoRI	SpeI
6	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
1	Plux-RBS-GFP-DT	EcoRI	SpeI
2	Plux-RBS-GFP-DT	EcoRI	SpeI
3	Plux-RBS-GFP-DT	EcoRI	SpeI
4	Plux-RBS-GFP-DT	EcoRI	SpeI
5	100bp ladder	--	--
6	apt12-1R-DT	XbaI	PstI
7	apt12-1R-DT	XbaI	PstI
8	apt12-1R-DT	XbaI	PstI
9	apt12-1R-DT	XbaI	PstI

Sep 11

Electrophoresis

Lane	Sample
1	1kb ladder
2	Pbad/araC+RBS-luxI-DT-1
3	Pbad/araC+RBS-luxI-DT-1
4	Plac+RBS-lysis-DT
5	Pcon+RBS-luxI-DT
6	1kb ladder

PCR

Tatsui

pKaiBC(200p)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
0.5	2.5	2.5	1.5	1.5	0.5	16	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

pKaiBC(400p)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
1	2.5	2.5	1.5	1.5	0.5	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

pKaiBC(800p)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
2	2.5	2.5	1.5	1.5	0.5	14.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

pKaiBC(1ng)	10*buffer KOD plus	dNTPs	MgSO4	primer mix(F&R)	KOD plus	MilliQ	total
2.5	2.5	2.5	1.5	1.5	0.5	14	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	60°C	68°C	30
2min	10sec	30sec	30sec

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	pKaiBC 800pg 60°C
3	pKaiBC 1ng 60°C
4	pKaiBC 800pg 61°C
5	pKaiBC 1ng 61°C
6	pKaiBC 800pg 62°C
7	pKaiBC 1ng 62°C
8	100bp ladder

colony PCR

Tatsui

Sample	base pair
9/9Pcon-lacZα(p3B4k5)	712bp
9/9Pcon-spinach-DT(p3B4k5)	605bp

Tatsui

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	42s	30

Sample	base pair
Pbad\araC-RBS-luxI-DT

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	Pcon-lacZα(pSB4K5)3
3	Pcon-lacZα(pSB4K5)4
4	Pcon-lacZα(pSB4K5)5
5	Pcon-lacZα(pSB4K5)6
6	Pcon-spinach-DT(pSB4K5)3
7	Pcon-spinach-DT(pSB4K5)4
8	Pcon-spinach-DT(pSB4K5)5
9	Pcon-spinach-DT(pSB4K5)6
10	100bp ladder

Lane	Sample
1	1kbp ladder
2	Pbad/araC-RBS-luxI-DT3
3	Pbad/araC-RBS-luxI-DT4
4	Pbad/araC-RBS-luxI-DT5
5	Pbad/araC-RBS-luxI-DT6
6	1kbp ladder

colony PCR

Sample	base pair
9/9Pcon+RBS-luxI-DT	1079bp
9/10Pcon+sptamer12_1R-DT	584bp
9/10DT+Pcon-pT181 attenuator 1	781bp
9/10Fusion1 attenuator(p3B1C3)-1	638bp

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
50sec	30sec	30sec	1min	30

Gel Extraction

Ligation

Okazaki

state	Vector		Inserter		Ligation High ver.2
experiment	9/8pSB4K5(EcoRI&SpeI)21.8ng/µL	2.3 µL	9/8Pcon-Spinach-DT(EcoRI&SpeI)10.5ng/µL	1.7 µL	2.0 µL
experment	9/8pSB4K5(EcoRI&SpeI)21.8ng/µL	2.3 µL	9/8RBS-lacZα-DT(EcoRI&SpeI)4.4ng/µL	6.7µL	3.5 µL
experiment	9/8pSB4K5(EcoRI&SpeI)21.8ng/µL	2.3 µL	9/8Pcon-RBS-lacZα-DT(EcoRI&SpeI)5.6ng/µL	1.9 µL	2.1 µL
experiment	9/6Plux 25ng/µL	2 µL	9/8 RBS-lysis1-DT(XbaI&PstI)	6.6 µL	3.5 µL
experiment	9/6Plux 25ng/µL	2 µL	9/8 RBS-lysis3-DT(XbaI&PstI)	4.7 µL	3.4 µL
experiment	9/10Pcon-pT181 attenuator(SpeI&PstI)	3.4µL	9/8RBS-lacZα-DT(EcoRI&SpeI)	4.5 µL	3.5 µL
experiment	9/6Pcon-RBS laxR-DT(EcoRI&XbaI) 26.7ng/µL	1.9 µL	Plax-RBS-GFP-DT	5.5 µL	3.5 µL
experiment	9/9 Pcon-pT181 attenuator(SpeI&PstI)	3.4 µL	9/10 aptamer12_1R-DT(XbaI&PstI)	1.7 µL	2.6 µL

Transformation

Nakamoto Tatsui

Name	Sample	Competent Cells	Total	Plate
9/9 Fusion1 antisense(pSB2C3)	2µL	20µL	22µL	CP
9/9 Fusion6 antisense(pSB2C3)	2µL	20µL	22µL	CP
9/9 aptamer 12_P(pSB2C3)	2µL	20µL	22µL	CP
9/9 aptamer 12_1&micro(pSB2C3)	2µL	20µL	22µL	CP
9/9 Fusion2 attenuator(pSB2C3)	2µL	20µL	22µL	CP
9/11 Pcon-spinach-DT(pSB4K5)	2µL	20µL	22µL	K
9/11 RBS-lacZα-DT(pSB4K5)	2µL	20µL	22µL	K
9/11 Pcon-RBS-lacZα-DT(pSB4K5)	2µL	20µL	22µL	K
Plux+RBS-lysis1-DT	2µL	20µL	22µL	CP
Plux+RBS-lysis3-DT	2µL	20µL	22µL	CP
Pcon-pT181attenuator+RBS-lacZα-DT	2µL	20µL	22µL	Amp
Pcon-iuxR+Plux-GFP	2µL	20µL	22µL	Amp
Pcon-pT181attenuator+apt12_1R-DT	2µL	20µL	22µL	Amp
Plac(BBa_R0011)	2µL	20µL	22µL	Amp

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	Pcon+RBS luxI-DT2
3	Pcon+RBS luxI-DT3
4	Pcon+RBS luxI-DT4
5	Pcon+RBS luxI-DT5
6	Pcon+RBS luxI-DT6
7	Pcon+apF12_1R-DT1
8	Pcon+apF12_1R-DT2
9	Pcon+apF12_1R-DT3
10	Pcon+apF12_1R-DT4
11	DT+Pcon-pT181attenuator1
12	F1attenuator(pSB1C3)
13	100bp ladder

Restriction Enzyme Digestion

Nakamoto

	DNA	EcoRI	XbaI	SpeI	PstI	buffer	BSA	MilliQ	Total
pSB4K5(EcoRI+SpeI)	9.4µL	1µL	0µL	1µL	0µL	3µL	3µL	12.6µL	30 µL
pSB4K5(EcoRI+SpeI) negative	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL
RBS-lacZα-DT(EcoRI+SpeI)	10µL	1µL	0µL	1µL	0µL	3µL	3µL	12µL	30µL
RBS-lacZα-DT(EcoRI+SpeI) negative	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL
Ptet-pT181 antisense(SteI+PstI)	10.8µL	0µL	0µL	1µL	1µL	3µL	3µL	12µL	30µL
Ptet-pT181 antisense(SteI+PstI) negative	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI)	7.1µL	1µL	0µL	1µL	0µL	3µL	3µL	14.9µL	30µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI) negative	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL
RBS-lysis2-DT(XbaI+PstI)	22µL	0µL	1µL	0µL	1µL	3µL	3µL	0µL	30µL
RBS-lysis2-DT(XbaI+PstI) negative	1.4µL	0µL	0µL	0µL	0µL	1µL	1µL	6.6µL	10µL

Liquid Culture

Nakamoto

Sample	medium
8/16 Master4 Plax
8/25 Pλ-laxI-2

PCR

Hirano

8/29 Pλ-RBS-luxI-DT(76.5ng/µL)	10x buffer for KOD plus-ver.2	KOD-plus	dNTP	MgSO4	F primer(RBS-luxI-DT-cloning-fwd)	R primer(RBS-luxI-DT-cloning-rev)	MilliQ	total
0.3	2.5	0.5	2.5	1.5	0.75	0.75	16.2	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	65°C	68°C	--
5min	30sec	30sec	1min	30

8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL)	10x buffer for KOD plus-ver.2	KOD-plus	dNTP	MgSO4	F primer(Pbad/araC(pSBlc3)-cloning-fwd)	R primer(Pbad/araC(pSBlc3)-cloning-rev)	MilliQ	total
0.3	2.5	0.5	2.5	1.5	0.75	0.75	16.2	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	54°C	68°C	--
5min	30sec	30sec	3min 20sec	30

Liquid Culture

Hirano

Sample	medium
9/4 RBS-lysis1+DT 3	plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis2+DT 1	plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis3+DT 3	plusgrow(+CP) 3ml 37°C

Sep 12

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
Pcon-pT181attenuator-DT	293.6	1.88	1.84
Pcon-apt12-1R-DT	303.8	1.75	1.64
Fusion1attenuator(pSB1c3)	172.0	1.96	2.31
RBS-lisis1-DT	235.1	1.59	1.60
RBS-lisis2-DT	387.7	1.91	2.01
RBS-lisis3-DT	387.9	1.91	2.09
Plux	166.7	1.98	1.63

Electrophoresis

Nakamoto

Lane	Sample
1	100bp ladder
2	9/11 P-RBS-lisis1-DT(Colony PCR prodution)

Liquid Culture

Nakamoto

Sample	medium
9/10 Pcon-apt12-1R-DT1	Plusgrow medium(+Amp)
9/10 DT-Pcon-pT181attenuator1	Plusgrow medium(+CP)
9/10 Fusion1-attenuator(pSB1c3)	Plusgrow medium(+CP)

Electrophoresis

Tatsui

Lane	Sample
1	1kb ladder
2	pSB4K5(EcoRI&SpeI)
3	pSB4K5 NC(EcoRI&SpeI)
4	RBS-lacz-DT(EcoRI&SpeI)
5	EcoRI&SpeI NC( EcoRI&SpeI)
6	Pcon-RBS-lacz-DT( EcoRI&SpeI)
7	Pcon-RBS-lacz-DT NC( EcoRI&SpeI)
8	1kb ladder

2

Lane	Sample
1	1kb ladder
2	Ptet-pT181antisense(SpeI&PstI)
3	Ptet-pT181antisense NC(SpeI&PstI)
4	RBS-lysis2-DT(XbaI&PstI)
5	EcoRI&SpeI NC( EcoRI&SpeI)
6	--
7	--
8	1kb ladder

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lacZα-DT	EcoRI&SpeI
3	RBS-lacZα-DT	EcoRI&SpeI
4	RBS-lacZα-DT	EcoRI&SpeI
5	--	--
6	Pcon-RBS-lacZα-DT	EcoRI&SpeI
7	Pcon-RBS-lacZα-DT	EcoRI&SpeI
8	Pcon-RBS-lacZα-DT	EcoRI&SpeI
9	1kb ladder	--

Name	concentration[µg/mL]	260/280	260/230
RBS-lacZα-DT(EcoRI&SpeI)	9.8	1.50	0.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)	11.2	1.47	0.08

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lysis2-DT	XbaI&PstI
3	RBS-lysis2-DT	XbaI&PstI
4	RBS-lysis2-DT	XbaI&PstI
5	--	--
6	1kb ladder	--

Name	concentration[µg/mL]	260/280	260/230
RBS-lysis2-DT(XbaI&PstI)	5.4	1.43	0.77

Electrophoresis

No name

Lane	Sample
1	1kb ladder
2	Pλ-RBS-luxI-DT(PCR product)
3	Pbad/araC-RBS-RFP(PCR product)

Colony PCR

Honda

Sample	base pair
9/11 apt12-1M(pSB1C3) 1	513
9/11 apt12-1M(pSB1C3) 2	513
9/11 Fusion3m2a09ttenuator(pSB1C3) 1	609
9/11 Fusion6 antisense(pSB1C3)1	431
9/11 aptamer 12-P(pSB1C3)1	515
9/11 Fusion1 antisense(pSB1C3) 1	420
9/11 Fusion1 antisense(pSB1C3) 2	420
9/11 Plac(BBa-R0011) 1	293
9/11 Plac(BBa-R0011) 2	293
9/11 Plac(BBa-R0011) 3	293
9/11 Plac(BBa-R0011) 4	293
9/11 Pcon-luxR-Plux-GFP 1	2138
9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1	712

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min12s	30cycles

Restriction Enzyme Digestion

Tatsui

	9/5 Pcon	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(PstI)	5.7	0µL	0µL	0µL	1µL	3µL	3µL	17.3µL	30µL
NC(PstI)	1.9	0µL	0µL	0µL	0µL	1µL	1µL	6.1µL	10µL

	8/17 DT	EcoRI	Buffer	BSA	MilliQ	total
1cut(EcoRI)	10.6	1µL	3µL	3µL	12.4µL	30µL
NC(EcoRI)	3.5	0µL	1µL	1µL	4.5µL	10µL

Colony PCR

Nakamoto and Tatsui

Sample	base pair
9/11 Pcon-pT181attenuator-RBS-lacZα-DT	965
9/11 Plux-PBS-lysis1-DT 1	613
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1	859
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2	859
9/11 Plux-RBS-lysis3-DT 1	1210
9/11 Plux-RBS-lysis3-DT 2	1210
Pcon-Spinach-DT(pSB4K5) 1	605

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min12s	30cycles

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/7 pSB4K5	2µL	20µL	Amp

Electrophoresis

No name

1

Lane	Sample
1	Fusion6 antisense
2	Fusion1 antisense-1
3	Fusion1 antisense-2
4	100bp ladder
5	Plac(BBa-R0011) 1
6	Plac(BBa-R0011) 2
7	Plac(BBa-R0011) 3
8	Plac(BBa-R0011) 4

2

Lane	Sample
1	1kb ladder
2	aptamer12-1M 1
3	aptamer12-1M 2
4	Fusion3m2 attenuator
5	aptamer12-P1
6	Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT
7	Pcon-RBS-lacZα=DT
8	1kb ladder

Electrophoresis

No name

Lane	Sample	Enzyme
1	Pcon-pT181attenuator-RBS-lacZα-DT 1	--
2	Pcon-pT181attenuator-RBS-lacZα-DT 2	--
3	Plux-RBS-lysis1-DT 1	--
4	Pcon-pT181attenuator-aptamer12-1R-DT 1	--
5	Pcon-pT181attenuator-aptamer12-1R-DT 2	--
6	Plux-RBS-lysis3-DT 1	--
7	1kbp ladder	--
8	Plux-RBS-lysis3-DT 2	--
9	Pcon-spinach-DT(pSB4K5) 1	--
10	9/5 Pcon	PstI
11	9/5 Pcon NC	--
12	8/17 DT	EcoRI
13	8/17 DT	--

Liquid Culture

Nakamoto

Sample	medium
9/11Fusion6 antisense-1	Plusgrow medium(+CP)
Plac(BBa-R0011)-3	Plusgrow medium(+Amp)
Plac(BBa-R0011)-4	Plusgrow medium(+Amp)
Fusion3m2 attenuator -1	Plusgrow medium(+CP)
Pλ-luxI-2	Plusgrow medium(+Amp)
9/11 aptamer12-1M-2	Plusgrow medium(+CP)

Colony PCR

Nakamoto

Sample	base pair
9/11 Fusion1 antisense 3	394
9/11 Fusion1 antisense 4	394
9/11 aptamer12-P 2	378
9/11 aptamer12-P 3	378

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	36s	30cycles

Electrophoresis

Nakamoto

Lane	Sample	Enzyme
1	Fusion1 antisense 2	--
2	Fusion1 antisense 3	--
3	1kb ladder	--
4	apt12-P 2	--
5	apt12-P 3	--

Restriction Enzyme Digestion

Tatsui

	9/12 RBS-lysis2-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.2	1.0µL	1.0µL	3.0µL	3.0µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/12 RBS-lysis3-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	5.2	1.0µL	1.0µL	3.0µL	3.0µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/11 RBS-lysis1-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	8.5	1.0µL	1.0µL	3.0µL	3.0µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	1.0µL	1.0µL	7.6µ	10µL

	8/20 J23100-RBS-luxR -DT	EcoRI	Buffer	BSA	MilliQ	total
1cut	5.8	1.0µL	3.0µL	3.0µL	17.2µL	30µL
NC	0.3µL	0µL	1.0µL	1.0µL	7.7µ	10µL

	9/10 pSB1C3	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	8.1	1.0µL	1.0µL	3.0µL	3.0µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1.0µL	1.0µL	7.6µ	10µL

	9/12 Plux	PstI	Buffer	BSA	MilliQ	total
1cut	12.0	1.0µL	3.0µL	3.0µL	11.0micro;L	30µL
NC	0.6µL	0µL	1.0µL	1.0µL	7.4µ	10µL

	9/10 pSB1C3	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	8.1	1.0µL	1.0µL	3.0µL	3.0µL	13.9µL	30µL

Electrophoresis

Tatsui

Lane	Sample	Enzyme1	Enzymel2
1	9/12 RBS-lysis2-DT	XbaI	PstI
2	9/12 RBS-lysis2-DT NC	--	--
3	9/11 RBS-lysis1-DT	XbaI	PstI
4	9/11 RBS-lysis1-DT NC	--	--
5	9/11 RBS-lysis3-DT	XbaI	PstI
6	9/11 RBS-lysis3-DT NC1	--	--
7	1kb ladder	--	--
8	100bp ladder	--	--
9	J23100-RBS-luxR-DT	EcoRI	--
10	J23100-RBS-luxR-DT	--	--
11	9/10 pSB1C3	EcoRI	SpeI
12	9/10 pSB1C3 NC	--	---
13	9/10 pSB1C3	XbaI	PstI
14	9/12 Plux	PstI	--
15	9/12 Plux NC	--	--

Observation through a confocal microscope

We used the liquid culture media with 200μL 9/10Pcon-pT181antisense-spinach-DT& 200μL 9/10 Pcon-spinach-DT& 200μL 9/10 JM109(overnight culture).
We translated them into each 1.5mL tube.

We elminated each supernatant using a 5000xg centrifuge for 1minute,.
Then,we resuspended with 100µL M9(distilled water) and eliminated each supernatant using a 5000xg centrifuge for 1 minute.x2

Adding 100µL M9(in 20µM DFHBI), we resuspended the pret.

15min after incubating in shield light, we eliminated each supernatant using a 5000xg centrifuge for 1 minute.

We looked at the fluorescence of the pret.
We failed to observe it.

Liquid Culture

Hirano

Sample	medium
9/10 Pcon-pT181antisense-spinach-DT	Plusgrow medium
9/10 Pcon-spinach-DT	Plusgrow medium
9/1 spinach-DT(RNA master plate)	Plusgrow medium

Sep 13

Miniprep

Nakamoto

DNA	concentration[µg/mL]	260/280	260/230
9/12 Fusion6 antisense	245.9	1.88	1.41
9/12 Plux-3	115.5	1.58	1.32
9/12 Plux-4	125.3	1.93	1.44
9/12 Fusion3n2 attenuator-1	231.9	1.81	1.31

Gel Extraction

Tatsui,Ashida

Lane	DNA	Enzyme
1	1kb ladder	--
2	RBS-lysis1-DT	XbaI&PstI
3	RBS-lysis1-DT	XbaI&PstI
4	RBS-lysis1-DT	XbaI&PstI

Lane	DNA	Enzyme
1	1kb ladder	--
3	RBS-lysis2-DT	XbaI&PstI
4	RBS-lysis2-DT	XbaI&PstI
5	RBS-lysis2-DT	XbaI&PstI

Lane	DNA	Enzyme
3	RBS-lysis3-DT	XbaI&PstI
4	RBS-lysis3-DT	XbaI&PstI
5	RBS-lysis3-DT	XbaI&PstI
6	1kb ladder	--

Lane	DNA	Enzyme
2	1kb ladder	--
3	pSB1C3	EcoRI&SpeI
4	pSB1C3	EcoRI&SpeI
5	pSB1C3	EcoRI&SpeI

Lane	DNA	Enzyme
1	1kb ladder	--
2	pSB1C3	XbaI&PstI
3	pSB1C3	XbaI&PstI
4	pSB1C3	XbaI&PstI

Restriction Enzyme Digestion

Nakamoto

	9/13 Plac	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(PstI)	16	0µL	0µL	0µL	1µL	3µL	3µL	7µL	30µL
NC	0.8	0µL	0µL	0µL	0µL	1µL	1µL	7.2µL	10µL

	9/10 Pcon attenuator	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(PstI)	10	0µL	0µL	0µL	1µL	3µL	3µL	13µL	30µL
NC	0.5	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/12 Plux	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(PstI)	12	0µL	0µL	0µL	1µL	3µL	3µL	11µL	30µL
NC	0.6	0µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/12 DT-E	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(XbaI)	23.7	0µL	0µL	1µL	0µL	3µL	3µL	0µL	30.7µL
NC	1.2	0µL	0µL	0µL	0µL	1µL	1µL	6.8µL	10µL

	9/12 Pcon-RBS-luxR-bTE	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(XbaI)	22.4	0µL	0µL	1µL	0µL	3µL	3µL	0.6µL	30µL
NC	2.2	0µL	0µL	0µL	0µL	1µL	1µL	5.8µL	10µL

	9/12 Pcon-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(SpeI)	21	0µL	1µL	0µL	0µL	3µL	3µL	12.0µL	30µL
NC	2.1	0µL	0µL	0µL	0µL	1µL	1µL	5.7µL	10µL

M9 (EDTA)

Kanbayashi

1mM EDTA

volume	10ml
Na2HPO4	300mg
KH2PO4	150mg
NaCl	25mg
NH4Cl	50mg
Fe(3)-EDTA	21mg

5mM EDTA

volume	10ml
Na2HPO4	300mg
KH2PO4	150mg
NaCl	25mg
NH4Cl	50mg
Fe(3)-EDTA	105mg

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	1kb ladder	--
3	Pcon-P	SpeI
4	Pcon-P	SpeI
5	Pcon-P	SpeI

Liquid Culture

Tatsui

Sample	medium
9/11 aptamer12_1M(pSB1C3)-1	--
9/11 aptamer 12_P1	--
9/11 Fusaion1 antisense-4	--
9/12 pSB4K5-1	--
9/12 pSB4K5-2	--

Restriction Enzyme Digestion

Nakamoto

	9/13 Plac-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(SpeI)	23.5	0µL	0.5µL	0µL	0µL	3µL	3µL	0µL	30µL
NC	8	0µL	0µL	0µL	0µL	1µL	1µL	0µL	10µL

	9/13 Pcon-pT181 attenuator-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cut(SpeI)	23.5	0µL	0.5µL	0µL	0µL	3µL	3µL	0µL	30µL
NC	02.3	0µL	0µL	0µL	0µL	1µL	1µL	5.7µL	10µL

	9/13 Plux-P	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
1cuts(SpeI)	23.8	0µL	0.2µL	0µL	1µL	3µL	3µL	0µL	30µL
NC	8	0µL	0µL	0µL	0µL	1µL	1µL	0µL	10µL

	9/5 RBS-lacZα-DT	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cut(XbaI&PstI)	10.8	0µL	0µL	1µL	1µL	3µL	3µL	11.2µL	30µL
NC	0.5	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

Electrophoresis

Hirano

Lane	Sample
1	100bp ladder
2	RBS-lacZα-DT(XbaI&PstI)
3	RBS-lacZα-DT(NC)

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Plac (SpeI&PstI)	8.5µL	9/13 pT181 antisense (XbaI&PstI)	2.1 µL	5.3 µL
experiment	9/13 Plac (SpeI&PstI)	8.5 µL	9/10 aptamer 12_1R-DT (XbaI&PstI)	1.8 µL	5.15µL
experiment	9/13 Plac (SpeI&PstI)	8.5 µL	9/2 pT181 attenuator(XbaI&PstI)	2.3 µL	10.4 µL
experiment	9/13 Plux (SpeI&PstI)	11.4 µL	9/13 RBS-lysis1-DT (XbaI&PstI)	5.0 µL	8.2 µL
experiment	9/13 Plux (SpeI&PstI)	11.4 µL	9/13 RBS-lysis2-DT (XbaI&PstI)	7.0 µL	9.2 µL
experiment	9/13 Plux (SpeI&PstI)	7.2 µL	9/13 RBS-lysis3-DT (XbaI&PstI)	6.0 µL	6.6 µL
experiment	9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)	2.0 µL	9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)	5.2 µL	3.6 µL
experiment	9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)	2.0 µL	9/10 aptamer 12_1R-DT (XbaI&PstI)	1.6 µL	1.8 µL
experiment	9/12 Ptet-pT181 antisense (--)	1.0 µL	9/10 spinach-DT (--)	2.4 µL	1.7 µL

PCR

No name

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/7 tetR aptamer 12_1R(pSB1C3)	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/6 pT181 antisense-1(pSB1C3)	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/6 pT181 attenuator (pSB1C3)	0.5	3.85	1.75	3.9	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/27 Pλ-RBS-luxI-DT-1	0.5	2.55	1.75	5.2	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/31 Pbad/araC-RBS-RFP-2	0.5	3.75	1.75	4	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/13 Plac(pSB1A2)	0.5	4.2	1.75	3.5	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/20 J23100-RBS-lacZα-DT	0.5	6.35	1.75	1.4	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/7 Ptet-RBS-lacZα-DT	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/8 J23100-spinach-DT	0.5	5.75	1.75	2	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/11 RBS-lysis1-DT	0.5	6.05	1.75	1.7	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/12 RBS-lysis2-DT	0.5	6.75	1.75	1.0	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/12 RBS-lysis3-DT	0.5	6.75	1.75	1.0	0.5	0	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/8 J23100-spinach-DT	0.5	6.35	1.75	1.4	0	0.5	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
8/27 Pλ-RBS-luxI-DT-1	0.5	2.55	1.75	5.2	0	0.5	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/12 RBS-lysis2-DT	0.5	6.75	1.75	1.0	0	0.5	10.5

genome DNA	Big Dye	MilliQ	5x buffer	temp(400mg)	SasA_fwd primer	SasA_rev primer	total
9/11 RBS-lysis1-DT	0.5	6.05	1.75	1.7	0	0.5	10.5

PreDenature	Denature	Annealing	Extension	cycle
96°C	96°C	50°C	68°C	--
2m	10s	5s	4m	30

PCR

Yoshida

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	Prefix-RBS-RpaB_fwd primer	RpaB-GG4-suffix_rev primer	MilliQ	total
RpaB	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	Prefix-PkaiBC_fwd primer ver.3	PkaiBC-suffix_rev primer ver.3	MilliQ	total
RpaB	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	58°C	68°C	--
2m	10s	30s	30s	30

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	100bp ladder	--
3	RBS-lacZα-DT	XbaI&PstI
4	RBS-lacZα-DT	XbaI&PstI
5	RBS-lacZα-DT	XbaI&PstI
6	RBS-lacZα-DT	XbaI&PstI

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/13 Plac+pT181 antisense	2µL	20µL	22µL	Amp
9/13 Plac+apt12_1R	2µL	20µL	22µL	Amp
9/13 Plac+pT181 attenuator	2µL	20µL	22µL	Amp
9/13 Plux+pT181 antisense	2µL	20µL	22µL	CP
9/13 Plux+RBS-lysis2-DT	2µL	20µL	22µL	CP
9/13 Plux+RBS-lysis2-DT	2µL	20µL	22µL	CP
9/13 Pcon-RBS-lacR-DT+Plux-RBS-GFP-DT	2µL	20µL	22µL	Amp
9/13 Pcon-pT181 antisense+apt12_1R-DT	2µL	20µL	22µL	Amp
9/13 Ptet-pT181antisense+spinach-DT	2µL	20µL	22µL	CP
Pλ-luxI-1	2µL	20µL	22µL	Amp

Sep 14

Restriction Enzyme Digestion

Hirano

	9/14 pSB4K5S	EcoRI	XbaI	SpeI	PstI	BufferB	BufferD	BSA	MilliQ	total
2cuts	10µL	1µL	0µL	1µL	0µL	3µL	0µL	3µL	12µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	0µL	1µL	7.5µL	10µL

	9/13 Plac	XbaI	PstI	BufferH	BSA	MilliQ	total
1 cut	9µL	0µL	1µL	3µL	3µL	14µL	30µL
NC	1µL	0µL	0µL	1µL	1µL	7µL	10µL

	9/14 PKaiBC	EcoRI	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	11µL	1µL	0µL	1µL	0µL	3µL	3µL	11µL	30µL
NC	4µL	0µL	0µL	0µL	0µL	1µL	1µL	4µL	10µL

	9/14 RpaB	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	13µL	0µL	1µL	0µL	1µL	3µL	3µL	9µL	30µL
NC	2µL	0µL	0µL	0µL	0µL	1µL	1µL	5µL	10µL

	8/17 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
1cut	9µL	0µL	1µL	0µL	0µL	3µL	3µL	14µL	30µL
NC	0.5µL	0µL	0µL	0µL	0µL	1µL	1µL	7.5µL	10µL

incubated 37°C 1hour

Liquid Culture

Hirano

Sample	medium
9/11 Plac(BBa-R0011)-(3)	Plusgrow medium(+Amp)

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	pSB4K5(1)	EcoRI	SpeI
3	pSB4K5(1)	--	--
4	Plac(1A2)	PstI	--
5	Plac(1A2)	--	--
6	PKaiBC	EcoRI	SpeI
7	PKaiBC	--	--
8	RpaB(1)	XbaI	PstI
9	RpaB(1)	--	--
10	RBS-GFP-DT	XbaI	--
11	RBS-GFP-DT	--	--
12	1kb ladder	--	--

Lane	Sample	Enzyme
1	1kb ladder	--
2	Plac(pSB1A2)	PstI
3	Plac(pSB1A2)	--

Sequence PCR

No name

genome DNA	Big Dye	5x buffer	temp(400mg)	primer	milliQ
Pcon-RBS-luxR-DT	0.5	1.75	0.9	0.5	6.85
Pcon-RBS-luxR-DT	0.5	1.75	0.4	0.5	7.35
Pcon-RBS-tetR-DT	0.5	1.75	3	0.5	5.25
Pcon-RBS-tetR-DT	0.5	1.75	3	0.5	5.25
Plux	0.5	1.75	2.4	0.5	5.35
Plux-RBS-GFP-DT	0.5	1.75	1.9	0.5	5.85
Plux-RBS-GFP-DT	0.5	0.5	1.9	0.5	5.85
Pcon-RBS-GFP-DT	0.5	0.5	1.2	0.5	6.55
Pcon-RBS-GFP-DT	0.5	0.5	1.2	0.5	6.55
RBS-lysis3-DT	0.5	0.5	1.0	0.5	6.75
Pcon-antisense-Spinach-DT	0.5	0.5	1.4	0.5	6.35
Pcon-RBS lacZα-DT	0.5	0.5	1.4	0.5	6.35
Ptet-RBS lacZα-DT	0.5	0.5	0.59	0.5	1.85

PreDenature	Denature	Annealing	Extension	cycle
96°C	96°C	50°C	60°C	--
2min	10sec	5sec	4min	30cycle

Gel Extraction

Tatsui

Lane	DNA	Enzyme
1	1kb ladder	--
2	pSB4K5	EcoRI&SpeI
3	pSB4K5	EcoRI&SpeI
4	pSB4K5	EcoRI&SpeI

Restriction Enzyme Digestion

No name

	9/14 Plac(1A2)	EcoRI	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	23µL	0µL	0µL	0.3µL	0µL	3µL	3µL	0µL	30µL

	9/14 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferH	BSA	MilliQ	total
2 cuts	23µL	1µL	0µL	0µL	0µL	3µL	3µL	0µL	30µL

Ligation

Nakamoto and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-PT181attenuator(SpeI&PstI)	1.7	9/13 RBS-lacZα-DT (XbaI & PstI)	12.5	5
experiment	9/13 pSB2C3 (XbaI & PstI)	1.9	9/14 RpaB (XbaI & PstI)	16.3	10.5
experiment	9/13 pSB2C3 (XbaI & PstI)	4.7	9/14 PkaiBC (EcoRI & SpeI)	6.7	5.7
experiment	9/14 RBS-GFP-DT (PstI&XbaI)	0.7	9/14 PkaiBC (EcoRI & SpeI)	2.6	1.6
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis1-DT (XbaI & PstI)	4.9	3.6
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis2-DT (XbaI & PstI)	7.0	4.7
experiment	9/14 Plac (SpeI&PstI)	2.4	9/14 RBS-lysis3-DT (XbaI & PstI)	9.2	5.8

Samples were evaporeted used evaporator into about 7 µL

Colony PCR

No name

Sample	base pair
9/13 Plux＋RBS-lysis1-DT (1)~(3)	--
9/13 Plux＋RBS-lysis2-DT (1)~(3)	-
9/13 Plux＋RBS-lysis3-DT (1)~(2)	-

Sample	base pair
9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)	--
9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)	-
9/13 Plac＋PT181attenuator (1)	-

Sample	base pair
Dλ-lux(1) (1)~(2)	--
NC	--

Electrophoresis

No name

Lane	Sample
1	1kbp ladder
2	9/13 Plux+RBS-lysis1-DT1
3	9/13 Plux+RBS-lysis1-DT2
4	9/13 Plux+RBS-lysis1-DT3
5	9/13 Plux+RBS-lysis2-DT1
6	9/13 Plux+RBS-lysis2-DT2
7	9/13 Plux+RBS-lysis2-DT3
8	9/13 Plux+RBS-lysis3-DT1
9	9/13 Plux+RBS-lysis3-DT2
10	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1
11	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2
12	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3
13	9/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4
14	9/13 Ptet-pT181 antisense+Spinach-DT1
15	9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
16	9/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
17	9/13 Plac+pT181 attenuator

Lane	Sample
1	1kbp ladder
2	Pλ-lux(1)1
3	Pλ-lux(1)1
4	NC

Liquid Culture

No name

Sample	medium
9/13 Pλ-luxI(1)-1	Plusgrow medium(+Amp)

Transformation

Nakamoto

Name	Sample	Competent Cells	Total	Plate
9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)	2µL	20µL	22µL	Amp
9/13 SB2C3＋9/14 PzαB	2µL	20µL	22µL	CP
9/13 pSB2C3＋9/14 PkaiBC	2µL	20µL	22µL	CP
9/13 RBS-GFP-DT＋9/14 PkaiBC	2µL	20µL	22µL	CP
Plac＋RBS-lysis1-DT	2µL	20µL	22µL	Amp
Plac＋RBS-lysis2-DT	2µL	20µL	22µL	Amp
Plac＋RBS-lysis2-DT	2µL	20µL	22µL	Amp

Sequence PCR

Tatsui

genome DNA	temp	Big Dye	5x buffer	primer	MilliQ
9/6 PT181 attenuator(VR)	3.9	0.5	1.75	0.5	3.85
8/17 RBS lacZα-DT	2.1	0.5	1.75	0.5	5.65
9/6 PT181 attenuator(VR)	3.9	0.5	1.75	0.5	3.85

PreDenature	Denature	Annealing	Extension	cycle
96°C	50°C	60°C	4°C	--
10sec	5sec	4min	1min	30cycle

Sep 15

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pλ-luxI	225	1.59	0.83

Liquid Culture

No name

Sample	medium
9/13 Pλ-luxI-1
9/7 pSB4K5-1
9/11 Plac-3

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon(SpeI & PstI)	3.6 µL	9/12 RBS-lacZα-DT(XbaI & PstI)	4.7 µL	8 µL

Restriction Enzyme Digestion

No Name

	DNA	EcorI	SpeI	Buffer	BSA	MilliQ	total
9/7Pcon-RBS-tetR-DT (EcoRI & SpeI)	15 µL	1 µL	1 µL	3 µL	3 µL	6 µL	30 µL
NC	0.7 µL	0 µL	0 µL	1 µL	1 µL	7.3 µL	10 µL

	DNA	EcoRI	SpeI	Buffer	BSA	MilliQ	total
9/12 Pcon-pT181 attenuator-DT (EcoRI & SpeI)	6.8 µL	1 µL	1 µL	3 µL	3 µL	15.2 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	EcoRI	SpeI	Buffer	BSA	MilliQ	total
9/12 Pcon-apt12_1R-DT(EcoRI & SpeI)	6.6 µL	1 µL	1 µL	3 µL	3 µL	15.4 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	EcoRI	SpeI	Buffer	BSA	MilliQ	total
9/8 Pcon-spinach-DT (EcoRI & SpeI)	7.6 µL	1 µL	1 µL	3 µL	3 µL	15.4 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/11 Pcon-pT181 anticodone-spinach-DT(XbaI & PstI)	7.2 µL	1 µL	1 µL	3 µL	3 µL	14.8 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
8/17 RBS-lacZα-DT (XbaI & PstI)	5.3 µL	1 µL	1 µL	3 µL	3 µL	16.7 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	EcoRI	XbaI	Buffer	BSA	MilliQ	total
9/7 Ptet-RBS-LacZα-DT (EcoRI & XbaI)	2.9 µL	1 µL	1 µL	3 µL	3 µL	18.5 µL	30 µL
NC	1.5 µL	0 µL	0 µL	1 µL	1 µL	7 µL	10 µL

Electrophoresis

No Name

Lane	Sample	Enzyme1	Enzyme2
1,14	100bp ladder
2	Pcon-RBS-tetR-DT	EcoRI	SpeI
3	Pcon-RBS-tetR-DT
4	Pcon-pT181 attenuator-DT	EcoRI	SpeI
5	Pcon-pT181 attenuator-DT
6	Pcon-apt12-1R-DT	EcoRI	SpeI
7	Pcon-apt12-1R-DT
8	Pcon-spinach-DT	EcoRI	SpeI
9	Pcon-spinach-DT
10	Pcon-pT181 antisense-spinach-DT	XbaI	PstI
11	Pcon-pT181 antisense-spinach-DT
12	RBS-lacZα-DT	XbaI	PstI
13	RBS-lacZα-DT
15	Ptet-RBS-lacZα-DT	EcoRI	XbaI
16	Ptet-RBS-lacZα-DT
17	1kbp ladder

Gel Extraction

No Name 1

Lane	DNA	Enzyme
1	100bp ladder
2~4	Pcon-pT181 attenuator-DT	EcoRI & SpeI
6~8	Pcon-apt12_1R-DT
10~12	Pcon-spinach-DT

2

Lane	DNA	Enzyme
1	100bp ladder
2~4	Pcon-pT181 anticodone-spinach-DT	XbaI & PstI
6~8	RBS-lacZα-DT	XbaI & PstI
10~12	Pcon-RBS-tetR-DT	EcoRI & SpeI

Name	concentration[µg/mL]	260/280	260/230
9/15 Ptet-RBS-lacZα-DT (EcoRI & SpeI)	1.9	1.08	0.77
9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI)	5.9	1.30	0.17
9/15 RBS-lacZα-DT (XbaI & PstI)	1.0	0.85	0.44
Pcon-pT181 anticodone-spinach-DT (XbaI & PstI)	1.3	0.95	0.28
Pcon-spinach-DT	0.9	1.37	0.05
Pcon-apt12_1R-DT	1.7	1.42	0.29
Pcon-pT181 attenuator-DT (EcoRI & SpeI)	2.1	1.53	0.46

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 pSB1C3 EcoRI & SpeI	4.7 µL	9/15 Pcon-RBS-tetR-DT EcoRI & SpeI	1.9 µL	xx µL
experiment	9/13 pSB1C3 EcoRI & SpeI	4.7 µL	9/15 Pcon-spinach-DT EcoRI & SpeI	4.7 µL	xx µL
experiment	9/13 pSB1C3 EcoRI & SpeI	4.7 µL	9/15 Pcon-apt12_1R-DT EcoRI & SpeI	2.0 µL	xx µL
experiment	9/13 pSB1C3 EcoRI & SpeI	4.7 µL	9/15 Pcon-pT181 attenuator-DT EcoRI & SpeI	3.0 µL	xx µL
experiment	9/13 pSB1C3 XbaI & PstI	1.9 µL	9/15 Pcon-pT181 anticodone-spinach-DT XbaI & PstI	4.4 µL	xx µL
experiment	9/13 pSB1C3 EcoRI & SpeI	4.7 µL	9/3 pT181 attenuator EcoRI & SpeI	3.8 µL	xx µL
experiment	9/6 Pcon SpeI & PstI	1.4 µL	9/15 RBS-lacZα-DT XbaI & PstI	2.0 µL	xx µL
experiment	9/14 pSB 4k5 EcoRI & SpeI	µL	9/15 Pcon-spinach-DT EcoRI & SpeI	9.4 µL	xx µL
experiment	9/15 Peet-RBS-lacZα-DT EcoRI & XbaI	2.6 µL	Pcon-RBS-tetR-DT EcoRI & SpeI	1.5 µL	xx µL

Restriction Enzyme Digestion

No Name

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/13 pT181 attenuator XbaI & PstI	6.3 µL	1 µL	1 µL	3 µL	3 µL	15.7 µL	30 µL
NC	0.3 µL	0 µL	0 µL	1 µL	1 µL	7.7 µL	10 µL

	DNA	EcoRI	SpeI	Buffer	BSA	MilliQ	total
9/10 pSB1C3 EcoRI & SpeI	8.1 µL	1 µL	1 µL	3 µL	3 µL	13.9 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	DNA	XbaI	PstI	Buffer	BSA	MilliQ	total
9/14 pSB4k5 XbaI & PstI	9.5 µL	1 µL	1 µL	3 µL	3 µL	12.5 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	SpeI	PstI	Buffer	BSA	MilliQ	total
9/11 DT SpeI & PstI	10.4 µL	1 µL	1 µL	3 µL	3 µL	11.6 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

	DNA	SpeI	PstI	Buffer	BSA	MilliQ	total
9/6 apt12_1R-DT SpeI & PstI	7.9 µL	1 µL	1 µL	3 µL	3 µL	14.1 µL	30 µL
NC	0.4 µL	0 µL	0 µL	1 µL	1 µL	7.6 µL	10 µL

	DNA	SpeI	PstI	Buffer	BSA	MilliQ	total
9/10 spinach-DT SpeI & PstI	9.9 µL	1 µL	1 µL	3 µL	3 µL	12.1 µL	30 µL
NC	0.5 µL	0 µL	0 µL	1 µL	1 µL	7.5 µL	10 µL

Liquid Culture

No name

Sample	medium
pSB1C3
DT
Spinach-DT

Sep 16

Liquid Culture

No name

Sample	medium
pSB1C3+P-KaiBC①	Plusgrow medium(+CP)
pSB1C3+P-KaiBC②	Plusgrow medium(+CP)

Colony PCR

No name

Sample	base pair
pSB1C3+P-KaiBC①	283
pSB1C3+P-KaiBC②	283

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
2min	30s	30s	18s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kb ladder
2	pT181 attenuator	XbaI+PstI
3	pT181 attenuator	XbaI+PstI
4	pT181 attenuator	XbaI+PstI
6	pSB1C3	EcoRI+SpeI
7	pSB1C3	EcoRI+SpeI
8	pSB1C3	EcoRI+SpeI
10	pSB4K5	XbaI+PstI
11	pSB4K5	XbaI+PstI
12	pSB4K5	XbaI+PstI

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator(XbaI+PstI)	8.2	1.62	0.11
pSB1C3(EcoRI+SpeI)	49.5	1.48	0.18
pSB4K5(XbaI+PstI)	18.1	1.73	0.57
DT(SpeI+PstI)	70.4	1.36	1.02
aptamer12-1R-DT(SpeI+PstI)	42.0	1.82	0.47
spinach-DT(SpeI+PstI)	36.8	1.79	0.61

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Dx-lux1	136.7	1.46	1.28
RBS-laczα-DT	237.6	1.88	1.70
pSB4K5	148.4	1.55	1.09
Pcon-KBS-tetR-DT	126.6	1.73	1.36
9/11Plac1	160.0	1.78	1.28
9/15Plac2	144.0	1.73	1.81
9/14Plac	332.5	1.51	2.33
Ptet-RBS-laczα-DT	231.3	1.84	1.57

PCR

No name

DNA	9/6pT181 antisense psB1C3(1)	KOD plus	10x buffer	dNTP	MgSO4	GG1_1C3_fwd primer	GG2_1C3_rev primer	MilliQ	total
400pg	1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
800pg	2	0.5	2.5	2.5	1.5	0.75	0.75	14.5	25
1000pg	2.5	0.5	2.5	2.5	1.5	0.75	0.75	14	25
nega	0	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	30s	30cycles

DNA	9/6 12-1R aptamer-DT	KOD plus	10x buffer	dNTP	MgSO4	GG1_1C3_fwd primer	GG1_1C3_rev primer	MilliQ	total
400pg	1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
800pg	2	0.5	2.5	2.5	1.5	0.75	0.75	14.5	25
1000pg	2.5	0.5	2.5	2.5	1.5	0.75	0.75	14	25
NC	0	0.5	2.5	2.5	1.5	0.75	0.75	16.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	30s	30cycles

Electrophoresis

Yoshida

Lane	Sample
1	100bp ladder
2	antisense 1000pg
3	antisense 800pg
4	antisense 400pg
5	tet aptamer-DT 1000pg
6	tet aptamer-DT 800pg
7	tet aptamer-DT 400pg
8	NC

Restriction Enzyme Digestion

Tatsui

	9/16 Pcon-RBS-tetR-DT(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	15.8	0	0	1	1	3	3	6.2	30
NC	0.8	0	0	0	0	1	1	7.2	10

	9/16 pSB4K5(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	13.5	0	0	1	1	3	3	8.5	30
NC	0.7	0	0	0	0	1	1	7.3	10

	8/22 pSB1C3(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	18.5	1	0	0	1	3	3	3.5	30
NC	0.9	0	0	0	0	1	1	7.1	10

	8/22 pSB1C3(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	18.5	1	1	0	0	3	3	3.5	30
NC	0.9	0	0	0	0	1	1	7.1	10

	8/21 pT181 attenuator2(µL)	EcoRI(µL)	SpeI(µL)	XbaI(µL)	PstI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	7.4	1	1	0	0	3	3	14.6	30
NC	0.4	0	0	0	0	1	1	7.6	10

Ligation

Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	Pcon-pT181antisense-spinach-DT(XbaI+PstI)	5.0µg/mL	pSB4K5(XbaI+PstI)	18.1µg/mL	xx µg/mL
experiment	pC181 attenuator(XbaI+PstI)	8.2µg/mL	pSB1C3(XbaI+PstI)	26.0µg/mL	xx µg/mL

Transformation

tatsui

Name	Sample	Competent Cells	Total
Pcon+RBS-laczα-DT	µL	µL	µL
Pcon-pT181 attenuator-DT+pSB1C3	µL	µL	µL
pT181 attenuator(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-aptamer12_1R-DT(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-spinach-DT(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-RBS-tetR-DT(EcoRI&SpeI)+pSB1C3	µL	µL	µL
Pcon-pT181 antisense-spinach-DT(XbaI&PstI)+pSB1C3	µL	µL	µL

Miniprep

tatsui

DNA	concentration[µg/mL]	260/280	260/230
9/15 pSBiC3	111.7	1.79	1.36
9/15 spinach-DT	113.9	1.57	1.38
9/15 DT	77.0	2.05	1.92

PCR

tatsui

9/14 aptamer12-P(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.5	0.5	1.75	0.5	5.75	10.5

9/14 aptamer12-1M(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.7	0.5	1.75	0.5	6.05	10.5

9/14 Fusion1 attenuator(VF)	Big Dye	5×Buffer	primer	MilliQ	total
2.3	0.5	1.75	0.5	5.45	10.5

9/14 Fusion1 antisense(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.3	0.5	1.75	0.5	6.45	10.5

9/13 Fusion3m2 attenuator(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.7	0.5	1.75	0.5	6.05	10.5

9/13 Fusion6 antisense(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.6	0.5	1.75	0.5	6.15	10.5

9/12 plux(VR)	Big Dye	5×Buffer	primer	MilliQ	total
2.4	0.5	1.75	0.5	5.35	10.5

9/9 Ptet antisense(VF)	Big Dye	5×Buffer	primer	MilliQ	total
2.2	0.5	1.75	0.5	5.55	10.5

9/12 Pcon-pT181 attenuator-DT(VF)	Big Dye	5×Buffer	primer	MilliQ	total
1.4	0.5	1.75	0.5	6.35	10.5

9/12 Pcon-pT181 attenuator-DT(VR)	Big Dye	5×Buffer	primer	MilliQ	total
1.4	0.5	1.75	0.5	6.35	10.5

PreDenature	Denature	Annealing	Extension	cycle
96°C	96°C	50°C	60°C	--
2min	10s	5s	4min	30cycles

Sep 17

Liquid Culture

Tatsui and Hirano

Sample	medium
Spinach-DT	LB medium(+CP)
Pcon-Spinach-DT	LB medium(+Amp)
Pcon-pT181 antisense-Spinach-DT	LB medium(+Amp)

PCR

Hirano

Pcon-tetR aptamer-DT(VF)	BigDye	5x buffer	primer(VF)	MilliQ	total
1.3	0.5	1.75	0.5	0.45	10.5

Pcon-tetR aptamer-DT(VR)	BigDye	5x buffer	primer(VR)	MilliQ	total
1.3	0.5	1.75	0.5	0.45	10.5

PreDenature	Denature	Annealing	Extension	cycle
96 °C	96 °C	50 °C	60 °C	--
2 min	10 sec	5 sec	4 min	30 cycles

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	Pcon-RBS-tetR-DT	XbaI	PstI
3	Pcon-RBS-tetR-DT	--	--
4	pSB4K5	XbaI	PstI
5	pSB4K5	--	--
6	pSB1C3	EcoRI	SpeI
7	pSB1C3	EcoRI	PstI
8	pSB1C3	--	--
9	pT181 attenuator	EcoRI	SpeI
10	pT181 attenuator	--	--
11	1kbp ladder	--	--

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	8/28 Plux (SpeI & PstI)	5.0 µL	9/13 RBS-lysis1-DT (XbaI & PstI)	3.5 µL	3.5 µL
experiment	8/28 Plux (SpeI & PstI)	5.0 µL	9/13 RBS-lysis2-DT (XbaI & PstI)	3.6 µL	3.5 µL
experiment	8/28 Plux (SpeI & PstI)	11.0 µL	9/13 RBS-lysis3-DT (XbaI & PstI)	9.5 µL	3.5 µL
experiment	9/14 Plac (SpeI & PstI)	2.4 µL	9/16 pT181 attenuator-DT (XbaI & PstI)	4.8 µL	3.5 µL
experiment	9/14 Plac (SpeI & PstI)	2.4 µL	9/3 pT181 antisense (XbaI & PstI)	2.0 µL	2.2 µL
experiment	9/14 Plac (SpeI & PstI)	2.4 µL	9/10 aptamer 12_1R-DT (XbaI & PstI)	1.8 µL	2.1 µL
experiment	9/13 pSB1C3 (XbaI & PstI)	2.0 µL	9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI)	12.0 µL	3.5 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-Spinach-DT (EcoRI & SpeI)	4.1 µL	2.5 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI)	2.0 µL	1.5 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI)	2.4 µL	1.7 µL
experiment	9/16 pSB1C3 (EcoRI & SpeI)	1.0 µL	9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI)	2.0 µL	1.5 µL
experiment	9/13 pSB1C3 (XbaI & PstI)	2.0 µL	9/16 Pcon-pT181-attenuator (XbaI & PstI)	4.4 µL	3.2 µL

Gel Extraction

No Name

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pT181 attenuator -(2)	EcoRI & SpeI
3
4

Name	concentration[µg/mL]	260/280	260/230
pT181 attenuator (EcoI & SpeI)	9.2	1.15	0.12

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB1C3	EcoRI & SpeI
3
4

Name	concentration[µg/mL]	260/280	260/230
pSB1C3	10.0	1.84	0.40

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB4K5	XbaI & PstI
3
4

Name	concentration[µg/mL]	260/280	260/230
pSB4K5	7.6	1.81	0.32

Lane	DNA	Enzyme
1	1kbp ladder	--
2	Pcon-RBS-tetR-DT	XbaI & PstI
3
4

Name	concentration[µg/mL]	260/280	260/230
Pcon-RBS-tetR-DT	8.0	2.40	1.14

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB1C3	EcoRI & PstI
3
4

Name	concentration[µg/mL]	260/280	260/230
pSB1C3	9.1	2.17	0.35

Mutation PCR

Kinose

pC KaiABC(100pg/µL)	Prime STAR(2x)	Primer(KaiA-BsaI MuT-Fwd)	Primer(KaiA-BsaI MuT-Rev)	MilliQ	total
1.0	25.0	1.0	1.0	22.0	50.0
1.5	25.0	1.0	1.0	21.5	50.0
2.0	25.0	1.0	1.0	21.0	50.0

PreDenature	Denature	Annealing	Extension	cycle
96 °C	98 °C	65 °C	72 °C	--
5 min	10 s	15 s	40 s	30 cycles

Sep 18

Restriction Enzyme Digestion

No name

	9/18Plux	SpeI	PstI	Buffer	BSA	MilliQ	total
2cuts	3.2µL	1µL	1µL	3µL	3µL	18.8µL	30µL
NC	1.7µL	0µL	0µL	1µL	1µL	6.3µL	10µL

	9/18Pcon-pT181antisense-spinach-DT②	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	7.5µL	1µL	1µL	3µL	3µL	14.5µL	30µL
NC	1.4icro;L	0µL	0µL	1µL	1µL	6.6µL	10µL

	9/16 Plac②	SpeI	PstI	Buffer	BSA	MilliQ	total
2cuts	13.8µL	1µL	1µL	3µL	3µL	14.5µL	30µL
NC	0.7µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/16 Spinach-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	17.6µL	1µL	1µL	3µL	3µL	4.4µL	30µL
NC	0.9µL	0µL	0µL	1µL	1µL	7.1µL	10µL

	9/8 Pcon J23100	SpeI	PstI	Buffer	BSA	MilliQ	total
2cuts	5.7µL	1µL	1µL	3µL	3µL	16.3µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/16 RBS-lacZα-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	8.4µL	1µL	1µL	3µL	3µL	13.6µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/11 Pcon-pT181antisense-spinach-DT&#9312:	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	7.2µL	1µL	1µL	3µL	3µL	14.8µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/6 apt12-1R-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	7.9µL	1µL	1µL	3µL	3µL	14.1µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/3 pT181antisense	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	6.3µL	1µL	1µL	3µL	3µL	15.7µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.7µL	10µL

	9/14 pSB4K5	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	7.3µL	1µL	1µL	3µL	3µL	14.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/31 Plux-RBS-GFP-DT	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	9.2µL	1µL	1µL	3µL	3µL	12.8µL	30µL
NC	0.5µL	0µL	0µL	1µL	1µL	7.5µL	10µL

	9/12 RBS-lysis3-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts	5.2µL	1µL	1µL	3µL	3µL	16.8µL	30µL
NC	0.3µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/18 Pcon-pT181attenuator	EcoRI	SpeI	Buffer	BSA	MilliQ	total
2cuts	14.3µL	1µL	1µL	3µL	3µL	7.7µL	30µL
NC	0.7µL	0µL	0µL	1µL	1µL	7.3µL	10µL

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	Plae②	SpeI	PstI
2	RBS-lysis3-DT	XbaI	PstI
3	Plux-RBS-GFP-DT	EcoRI	SpeI
4	PSB4K5	EcoRI	SpeI
5	Pcon	SpeI	PstI
6	Plux	SpeI	PstI
7	1kbp ladder	--	--
8	Plae②	--	--
9	RBS-lysis3-DT	--	--
10	Plux-RBS-GFP-DT	--	--
11	PSB4K5	--	--
12	Pcon	--	--
13	Plux	--	--

Electrophoresis

Okazaki

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon pT181antisense spinach-DT②	XbaI	PstI
3	Spinach-DT	XbaI	PstI
4	RBS-lacZα-DT	XbaI	PstI
5	Pcon pT181antisense-Spinach-DT①	XbaI	PstI
6	aptamer12-1R-DT	XbaI	PstI
7	pT181attenuator	XbaI	PstI
8	Pcon attenuator	EcoRI	SpeI

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon pT181antisense spinach-DT②	XbaI	PstI
3	Spinach-DT	XbaI	PstI
4	RBS-lacZα-DT	XbaI	PstI
5	Pcon pT181antisense-Spinach-DT①	XbaI	PstI
6	apt12-1R-DT	XbaI	PstI
7	pT181attenuator	XbaI	PstI
8	Pcon attenuator	EcoRI	SpeI

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon pT181antisense spinach-DT②	--	--
3	Spinach-DT	--	--
4	RBS-lacZα-DT	--	--
5	Pcon pT181antisense-Spinach-DT①	--	--
6	apt12-1R-DT	--	--
7	pT181attenuator	--	--
8	Pcon attenuator	--	--

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ldder	--
2	Plac②	SpeI+PstI
3	Plac②	SpeI+PstI
4	--	--
5	RBS-lysis3-DT	XbaI+PstI
6	RBS-lysis3-DT	XbaI+PstI
7	--	--
8	Plux-RBS-GFP-DT	EcoRI+SpeI
9	Plux-RBS-GFP-DT	EcoRi+SpeI
10	--	--
11	PSB4KS	EcoRI+SpeI
12	PSB4KS	EcoRI+SpeI

Lane	DNA	Enzyme
1	1kbp ldder	--
2	Pcon	SpeI+PstI
3	Pcon	SpeI+PstI
4	--	--
5	Plux	SpeI+PStI
6	Plux	SpeI+PStI

Lane	DNA	Enzyme
1	Pcon PT181 antisense spinach-DT	XbaI+PstI
2	Pcon PT181 antisense Spinach-DT	XbaI+PstI
3	100bp ladder	--
4	Pcon PT181 antisense Spinach-DT	XbaI+PstI
5	Pcon PT181 antisense Spinach-DT	XbaI+PstI
6	1kbp ladder	--

Lane	DNA	Enzyme
1	100bp ladder	--
2	apt12-1R-DT	XbaI+PstI
3	apt12-1R-DT	XbaI+PstI
4	--	--
5	PT181 attenuator	XbaI+PstI
6	PT181 attenuator	XbaI+PstI

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pcoa attenuator	EcoRI+PstI
3	Pcoa attenuator	EcoRI+PstI
4	--	--
5	RBS-lacZα-DT	XbaI+PstI
6	RBS-lacZα-DT	XbaI+PstI

Sep 19

Sep 20

Ligation

Okazaki, Kanaoka, Tatsui and Hirano

state	Vector		Inserter		Ligation High ver.2
experiment	9/18 Plac (SpeI & PstI)	3.0 µL	9/18 RBS-lysis3-DT (XbaI & PstI)	12.4 µL	3.5 µL
experiment	9/12 Ptet-pT181 antisense	1.0 µL	9/18 spinach-DT	11.9 µL	3.5 µL
experiment	9/13 Pcon-RBS-luxR-DT	2.1 µL	9/10 Plux-RBS-GFP-DT	5.6 µL	3.5 µL
experiment	9/13 Pcon-pT181 attenuator (SpeI & PstI)	2.0 µL	9/18 aptamer 12-1R-DT (XbaI & PstI)	6.3 µL	3.5 µL
experiment	9/18 Plac ②(SpeI & PstI)	3.0 µL	9/18 aptamer 12-1R-DT(XbaI & PstI)	4.5µL	x3.5 µL

Transformation

Tatsui

Name	Sample	Competent Cells	Plate
9/17 pSB1C3-Pcon-pT181 attenuator-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-antisense-spinach-DT	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-RBS-tetR-DT x2	2 µL	20 µL	CP
9/14 pSB1C3-PkaiBC	2 µL	20 µL	CP
9/14 pSB1C3-RpaB	2 µL	20 µL	CP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	CP
9/17 pSB1C3-Pcon-spinach-DT	2 µL	20 µL	CP
9/14 RBS-GFP-DT-PkaiBC	2 µL	20 µL	CP
Plux-RBS-lysis3-DT	2 µL	20 µL	CP
9/20 Ptet-pT181 antisense-spinach-DT	2 µL	20 µL	CP
9/20 Pcon-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5)	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 KaiABC	2 µL	20 µL	Kan
9/20 Plac-RBS-lysis3-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer 12_1R	2 µL	20 µL	Amp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT	2 µL	20 µL	Amp
Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20 Plac-aptamer12_1R-DT	2 µL	20 µL	Amp
9/20Pcon-pT181attenuator-aptamer-DT	2 µL	20 µL	Amp
9/17 Plac-pT181-attenuatorx2	2 µL	20 µL	Amp
9/15 Pcon-RBS-lacZα-DT	2 µL	20 µL	Amp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT	2 µL	20 µL	Amp
PUC19	2 µL	20 µL	Amp

PCR

Tatsui

Sample	base pair
pT181 attenuator(pSB1C3)	--
Pcon-RBS-lacZα-DT	--

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 s	30 s	3 min	30 cycles

Electrophoresis

No name

Lane	Sample	Enzyme
1	Pcon-RBS-lacZα-DT	--
2	1kb ladder	--
3	pSB1C3-pT181attenuator	--

Sep 21

Electrophoresis

no name

Lane	Sample	Enzyme1	Enzyme2
1	Pcon-RBS-lacZα-DT	--	--
2	1kbp ladder	--	--
3	pSB1C3+pT181 attenuator	--	--

Liquid Culture

No name

Sample	medium
9/2 spinach-DT-2	--
8/16 Plux-4	--
8/27 Plux-RBS-GFP-DT-1	--
9/5 Pcon-pT181 attenuator-1	--

Master Plate

No name

Number	Use LB plate
1	8/27 Pλ-luxI-1(+Amp)
2	9/10 Plac(BBa-R0011)-3(+Amp)
3	9/5 Pcon-RBS-tetR-DT-1(+Amp)
4	9/5 Pcon-pT181 antisense-1(+Amp)
5	9/15 Pcon-pT181 attenuator(+Amp)
6	9/- Pcon-apt12_1R-DT-1(+Amp)
7	9/5 Pcon-spinach-DT-3(+Amp)
8	8/27 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(+Amp)

Number	Use LB plate
1	9/4 pT181 antisense(pSB1C3)-1(+CP)
2	9/11 Fusion1 antisense(pSB1C3)-4(+CP)
3	9/11 Fusion6 antisense(pSB1C3)-1(+CP)
4	9/4 pT181 attenuator(pSB1C3)-1(+CP)
5	9/10 Fusion1 attenuator(pSB1C3)-1(+CP)
6	9/11 Fusion3n2 attenuator(pSB1C3)-1(+CP)
7	9/4 aptamer12_1R(pSB1C3)-1(+CP)
8	9/11 aptamer12_P(pSB1C3)-3(+CP)
9	9/11 aptamer12_1M(pSB1C3)-1(+CP)

Number	Use LB plate
1	8/29 Pbad/araC-RBS-RFP-DT-1(+CP)
2	9/4 RBS-lysis3-DT-3(+CP)
3	9/10 Pcon-pT181 attenuator-DT-1(+CP)
4	9/5 Ptet-pT181 antisense-1(+CP)
5	9/4aptamer 12_1R-DT-1(+CP)
6	9/5 Ptet-RBS-lacZα-DT-4(+CP)
7	8/10 RBS-lacI-DT-2(+CP)
8	8/29 Plux-RBS-GFP-DT-1(+CP)

Restriction Enzyme Digestion

Noname

	8/17 RBS-DT	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts(XbaI&PstI)	8.1	0µL	0µL	1µL	1µL	3µL	3µL	13.9µL	30µL
NC	0.4	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	8/17 RBS-DT	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cut(EcoRI&XbaI)	16.2	1µL	1µL	0µL	0µL	3µL	3µL	5.8µL	30µL

	9/15 Pcon-pT181attenuator	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cuts(EcoRI&PstI)	12	1µL	0µL	0µL	1µL	3µL	3µL	10µL	30µL
NC	0.6	0µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	8/20 Pcon=RBS-GFP-DT-1	EcoRI	SpeI	XbaI	PstI	Buffer	BSA	MilliQ	total
2cut(EcoRI&XbaI)	6	1µL	0µL	1µL	0µL	3µL	3µL	0µL	14µL
NC	0.3	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

incubate 37°C 1hour

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT	XbaI	PstI
2	RBS-GFP-DT	--	--
3	RBS-GFP-DT	EcoRI	XbaI
4	1kbp ladder	--	--
5	Pcon-pT181attenuator	SpeI	PstI
6	Pcon-pT181attenuator	--	--
7	Pcon-RBs-GFP-DT	EcoRI	XbaI
8	Pcon-RBs-GFP-DT	--	--

PCR

Nakamoto

Sample	base pair
Pcon-apt12_1R-DT(E-1A)	50
Pcon-spinach-DT(E-1A)	402
Pcon-pT181 attenuator(E-1A)	400
DT(1-3)	65
tetR aptamer 12_1R-DT(1-3)	283
Pcon-pT181 attenuator(1-SA)	303
spinach-DT(2-1)	338
Pcon-pT181 attenuator(E-0)	403
Pcon-pT181 attenuator(2-SA)	403
spinach-DT(3-2)	395
Ptet-PT181 anisense	400

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	35s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kb ladder	--
3	RBS-GFP-DT	XbaI&PstI
4	RBS-GFP-DT	XbaI&PstI
5	RBS-GFP-DT	XbaI&PstI

Name	concentration[µg/mL]	260/280	260/230
RBS-GFP-DT(XbaI&PstI)	14.8	1.74	0.47
RBS-GFP-DT(EcoRI&SpeI)	88.4	1.85	1.49
Pcon-RBS-GFP-DT(EcoRI&XbaI)	47.5	1.82	1.12

Sep 22

Colony PCR

Tatsui

Sample	base pair
9/20 Plac-PT181 attenuator(1~4)	664
9/20 Plac-PT181 antisense(1~6)	369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)	859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)	723

Electrophoresis

Nakamoto

Lane	Sample
1	Pcon-apt12_1R-DT(E-1A)
2	Pcon-spinach-DT(E-1A)
3	Pcon-pT181 attenuator(E-1A)
4	DT(1-3)
5	tetR aptamer 12_1R-DT(1-3)
6	100bp ladder
7	Pcon-pT181 attenuator(1-SA)
8	spinach-DT(2-1)
9	Pcon-pT181 attenuator(E-0)
10	Pcon-pT181 attenuator(2-SA)
11	spinach-DT(3-2)
12	Ptet-PT181 anisense

Miniprep

Honda

DNA	concentration[µg/mL]	260/280	260/230
9/21 spinach-DT	197.9	1.67	1.54
9/21 Plux	125.9	1.71	1.27
9/21 Plux-RBS-GFP-DT-(1)	168.0	1.62	1.73
9/21 Pcon-PT181 attenuator-(1)	368.3	1.57	1.62
9/21 RBS-GFP-DT	532.8	1.75	1.78
9/21 Pcon-RBS-GFP-DT	845.2	2.02	1.70

Colony PCR

Honda

Sample	base pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)	283

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min10s	30cycles

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-aptamer12-1R-DT(E-1A)	--
6~8	Pcon-spinach-DT(E-1A)	--
10~12	Pcon-attenuator PT181(E-1A)	--

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	DT(1-3)	--
6~8	aptamer12-1RDT(1-3)	--
10~12	Pcon-PT181 attenuator(1-5A)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-aptamer12-1R-DT(E-1A)	31.2	1.87	0.30
Pcon-spinach-DT(E-1A)	25.6	1.88	0.99
Pcon-attenuator PT181(E-1A)	25.3	1.99	0.86
DT(1-3)	22.1	1.82	0.20
aptamer12-1RDT(1-3)	33.5	1.99	0.05
Pcon-PT181 attenuator(1-5A)	36.9	1.12	0.96

Restriction Enzyme Digestion

No name

	9/16 Plac	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	12.5µL	0µL	1µL	1µL	3µL	3µL	9.5µL	30µL
NC	0.8µL	0µL	0µL	0µL	1µL	1µL	7.4µL	10µL

	9/22 Plux	XbaI	SpeI	PstI	BufferB	BSA	MilliQ	total
2cuts	16µL	0µL	1µL	1µL	3µL	3µL	14µL	30µL
NC	0.8µL	0µL	0µL	0µL	1µL	1µL	7.2µL	10µL

	9/20 RBS-tetR-2R-DT	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	8.5µL	1µL	0µL	1µL	3µL	3µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	0µL	1µL	1µL	7.6µL	10µL

Gel Extraction

No name

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-attenuator PT181(E-0)	--

Lane	DNA	Enzyme
1	100bp ladder	--
2~4	Pcon-attenuator PT181(E-0)	--

Lane	DNA	Enzyme
2	100bp ladder	--
4~6	Pcon-attenuator PT181(2-SA)	--

Lane	DNA	Enzyme
1	100bp ladder	--
3~5	spinach-DT(3-2)	--
8~10	Ptet-antisense PT181(1-3)	--

Name	concentration[µg/mL]	260/280	260/230
Pcon-attenuator PT181(E-0)	2.4	1.80	0.17
Pcon-attenuator PT181(2-SA)	10.8	1.85	0.54
spinach-DT(3-2)	36.7	1.80	0.75
Ptet-antisense PT181(1-3)	16.8	1.74	0.78

Colony PCR

No name

Sample	base pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)	859

Colony PCR

Tatsui

Sample	base pair
PconPT181antisense-spinach-DT(9~15)	723

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-PT181 attenuator (SpeI & PstI)	1.7 µL	9/18 aptamer-1R-DT (XbaI & PstI)	5.4µL	3.5µL
experiment	9/12 Plac(SpeI & PstI)	2.4 µL	9/18 aptamer-1R-DT (XbaI & PstI)	6.1µL	3.5 µL
experiment	9/13 Ptet(SpeI & PstI)	1.7 µL	9/10 Plux-RBS-GFP-DT	7.0 µL	4.4 µL
experiment	9/21 RBS-GFP-DT (EcoRI & XbaI)	0.6 µL	9/18 aptamer 12-1R-DT (XbaI & PstI)	5.6µL	3.1 µL
experiment	9/13 PSB1C3(XbaI & PstI)	1.9 µL	9/16 PT181 attenuator(XbaI & PstI)	4.6µL	3.3 µL

Liquid Culture

No name

Sample	medium
9/20 Plac-antisense-1	--
9/20 Plac-attenuator-1	--
9/20 Plac-antisense-1	--
9/20 Plac-attenuator-1	--

digestion

No name

	9/8 Pcon-PT181 antisense	EcoRI	SpeI	BufferM	MilliQ	total
2cuts	8.3µL	1µL	1µL	3µL	16.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	8.3µL	10µL

	9/7 Pcon-RBS-tetR-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cut	14µL	1µL	1µL	0µL	0µL	3µL	3µL	18.2µL	30µL
NC	0.7µL	0µL	0µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/22 RBS-GFP-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ	total
2cuts	3.8µL	1µL	1µL	0µL	0µL	3µL	3µL	18.2µL	30µL
NC	0.2µL	0µL	0µL	0µL	0µL	1µL	1µL	7.8µL	10µL

	9/6 aptamer12-1R-DT	EcoRI	XbaI	SpeI	PstI	BufferD	BSA	MilliQ
2cut	8µL	1µL	1µL	0µL	0µL	3µL	3µL	16µL
NC	0.4µL	0µL	0µL	0µL	0µL	1µL	1µL	7.6µL

incubate 37°C 1hour

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3~5	Pcon-PT181 antisense (EcoRI&SpaI)	--

Transformation

No name

Name	Sample	Competent Cells	Plate
9/20 Plac-aptamer12-1R-DT(XbaI&PstI)	2 µL	20 µL	Amp
9/20 pT181 attenuator(1C3)(EcoRI&SpeI)	2 µL	20 µL	CP
9/17 pT181 attenuator(1C3)(XbaI&PstI)	2 µL	20 µL	CP
9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI)	2 µL	20 µL	Amp
kaiABC	2 µL	20 µL	Amp
9/22 pSB1C3-RpaB(XbaI&PstI)	2 µL	20 µL	Amp
9/22 pSB1C3-pT181 attenuator(XbaI&PstI)	2 µL	20 µL	Amp
9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI)	2 µL	20 µL	CP
9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI)	2 µL	20 µL	CP
9/22 pT181 attenuator(XbaI&PstI)(1C3)	2 µL	20 µL	CP

PCR

No name

Sample	base pair
Pcon-RBS-tetR-DT(1-2(A))	--
Pcon-RBS-tetR-DT(3-2(A))	--
Pcon-RBS-GFP-DT(1-S(A))	--
Pcon-RBS-GFP-DT(2-S(A))	--
Pcon-tetR-DT(E-2(A))	--

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	57 °C	68 °C	--
2 min	10 s	30 s	1min5s	30 cycles

Electrophoresis

No name

Lane	Sample
1	1kbp ladder
2	Pcon-RBS-tetR-DT(1-2Ⓐ)
3	Pcon-RBS-tetR-DT(3-2Ⓐ)
4	Pcon-RBS-GFP-DT(1-SⒶ)
5	Pcon-RBS-GFP-DT(2-SⒶ)
6	Pcon-tetR-DT(E-2Ⓐ)

Sep 23

Transformation

No name

Name	Sample	Competent Cells	Plate
9/14 pSB1C3＋PkaiBC	2 µL	20 µL	--
9/14 pSB1C3＋RpaB	2 µL	20 µL	--
9/14 pkaiBC＋Rbs-GFP-DT	2 µL	20 µL	--

Gel Extraction

No Name

Lane	DNA	Enzyme
1	1kbp ladder
3-4	Pcon-RBS-tetR-PT

Name	concentration[µg/mL]	260/280	260/230
Pcon-PT181 anti EcoRI+SpeI	46.7	1.06	0.93
Pcon-RBS-tetR-DT EcoRI+XbaI	119.3	0.99
RBS-8FP-DT EcoRI+XbaI	87.7	1.00	0.98
ap42_1R-DT EcoRI+XbaI	68.8	1.32	1.03
Pcon-RBS-tetR-DT 1-2 A	203.0	1.66	1.79
Pcon-RBS-tetR-DT 3-2 A	67.7	2.64	1.28
Pcon-RBS-GFP-DT 1-S A	185.6	1.88	2.04
Pcon-RBS-GFP-DT 2-S A	298.6	1.68	1.55
Pcon-tetR-DT E-2 A	202.4	1.57	1.66

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
9/8 Pcon-pT181 anti	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
8/20 Ptet(1)	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25
9/16 Plac 2	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94 °C	98 °C	57 °C	68 °C	--
2min	10sec	30sec	20sec	30cycles

Ligation

Yoshida

state	Vector		Inserter		Ligation High ver.2
experiment	9/21 Pcon-RBS-GFP-DT EcoRI+XbaI	47.5 µg/mL	9/15 Pcon-RBS-tetR-DT EcoRI+SpeI	59 µg/mL	2.3 µg/mL
experiment	9/21 Pcon-RBS-GFP-DT EcoRI+XbaI	47.5 µg/mL	9/15 Pcon-apt12_1R-DT EcoRI+SpeI	17 µg/mL	2.4 µg/mL
experiment	9/21 Pcon-RBS-GFRDT EcoRI+XbaI	2.1 µg/mL	9/15 Pcon-DT181 attenuator-DT	21 µg/mL	3.0 µg/mL
experiment	9/22 apt 12_1R-DT EcoRI+XbaI	67.8 µg/mL	9/18 Pcon-DT181 attenuator EcoRI+SpeI	5.7 µg/mL	3.5 µg/mL
experiment	9/22RBS-GFP-DT EcoRI+XbaI	87.7 µg/mL	9/18 Pcon-DT18 attenuator EcoRI+SpeI	6.7 µg/mL	3.5 µg/mL
experiment	9/13 DT EcoRI+XbaI	25.4 µg/mL	9/22 Pcon-DT181 antisense EcoRI+SpeI	46.8 µg/mL	2.3 µg/mL

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	SasA_fwd primer	SasA_rev primer	MilliQ	total
Pcon-attenuator-DT (E-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-attenuator-DT (2-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-antisense-spinach-DT (1-2A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25
Pcon-antisense-spinach-DT (E-1A)	0.5	2.5	2.5	1.5	0.75	0.75	77.5	25

PreDenature	Denature	Annealing	Extension	cycle
94 °C	98 °C	57 °C	68 °C	--
2min	10s	30s	45s	30cycle

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/22 Plac-DT181 attenuator-1	204.0	2.00	1.95
9/22 Plac-DT181 attenuator-2	195.3	1.84	1.62
9/22 Plac-DT181 antisense-1	188.2	1.87	2.15
9/22 Plac-DT181 antisense-2	165.0	1.95	2.66

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1,6	100bp ladder(Promega)	--	--
2	Pcon-attenuator-DT (E-1A)	--	--
3	Pcon-attenuator-DT (2-1A)	--	--
4	Pcon-antisense-spinach-DT (1-2A)	--	--
5	Pcon-antisense-spinach-DT (E-1A)	--	--

Gel Extraction

Hirano

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator-DT(E-1A)	166.7	1.51	0.99
Pcon-pT181 attenuator-DT(2-1A)	122.1	1.75	1.20
Pcon-pT181 antisense-spinach-DT(1-2A)	99.1	1.86	1.81

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	9/8 Pcon-pT181 antisense(E-1A)	--	--
3	8/20 Ptet(1)(2-S)	--	--
4	9/16 Plac2(E-1A)	--	--

Transformation

No Name

Name	Sample	Competent Cells	Total	Plate
pSB1C3	2µL	20µL	22µL

Ligation

No Name

state	Vector		Inserter		Ligation High ver.2
experiment	9/19 Plac SpeI+PstI	24.6 µg/mL	9/18 ape12_1R-DT XbaI+PstI	4.5 µg/mL	2 µg/mL
experiment	9/21 RBS-GFP-DT EcoRI+XbaI	88.4 µg/mL	9/15 Pcon-pT181 attenuator EcoRI+SpeI	21 µg/mL	2 µg/mL
experiment	9/13 PSB1C3 XbaI+PstI	26.0 µg/mL	9/16 pT181 attenuator XbaI+PstI	8.2 µg/mL	2 µg/mL

Colony PCR

Tatsui

Sample	base pair
9/22 Pcon-PT181 attenuator12-1R-DT(1~8)	--
9/22 Pcon-PT181 attenuator(1~4)	--
9/20 Plac＋aptamer12-1R-DT(1~4)	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	54s	30cycle

Sep 24

Electrophoresis

No name

Lane	Sample
1	9/20 Pcon-pT181aptamer12-1R-DT 1
2	9/20 Pcon-pT181aptamer12-1R-DT 2
3	9/20 Pcon-pT181aptamer12-1R-DT 3
4	9/20 Pcon-pT181aptamer12-1R-DT 4
5	9/20 Pcon-pT181aptamer12-1R-DT 5
6	9/20 Pcon-pT181aptamer12-1R-DT 6
7	9/20 Pcon-pT181aptamer12-1R-DT 7
8	9/20 Pcon-pT181aptamer12-1R-DT 8
9	100bp ladder
10	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 1
11	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 2
12	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 3
13	9/22 Pcon-pT181attenuator-aptamer12-1R-DT 4
14	9/20 Plac-aptamer12-1R-DT 1
15	9/20 Plac-aptamer12-1R-DT 2
16	9/20 Plac-aptamer12-1R-DT 3
17	9/20 Plac-aptamer12-1R-DT 4

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 pSB1C3-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
RBS-GFP-DT+Pcon-pT181attenuator	2 µL	20 µL	22 µL	LB (+CP)
9/23 Plac-+spinach-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+Amp)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS-GFP-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
PUC19	2 µL	20 µL	22 µL	LB (+Amp)
Ptet-RBS-lacZα-DT	2 µL	20 µL	22 µL	LB (+CP)
pSB1C3	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
aptamer12-1R-DT(Master plate)	LB(+CP)

Restriction Enzyme Digestion(PCR product)

Hirano

Concentrate PCR product by evaporator until volume become less than 1µL, add all sulution and pipetting.

	10*cut smart buffer	BsaI-HF	MilliQ	total
9/22 Pcon-pT181 attenuator (E-0)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-pT181 attenuator (2-3A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 spinach-DT(3-2)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Ptet-pT181 antisense (1-3)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-tet aptamer-DT (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-spinach-DT (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-pT181 attenuator (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 DT (1-3)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 tet aptamer-DT (1-3)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-pT181 attenuator (1-SA)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-tetR-DT (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-tetR-DT (3-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-tetR-DT (E-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-GFP-DT (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/22 Pcon-RBS-GFP-DT (2-SA)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 attenuator-DT (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 attenuator-DT (0-1A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 antisense-spinach-DT (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Pcon-pT181 antisense (1-2A)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Ptet-1 (2-S)	2.0 µL	0.3 µL	17 µL	19.3µL
9/23 Plac-2 (E-1A)	2.0 µL	0.3 µL	17 µL	19.3µL

Incubate 37°C 12hour

Column Refining

Kojima

Name	concentration[µg/mL]	260/280	260/230
8/22 Pcon-pT181 attenuator(E-0)	7.8	1.70	0.03
8/22 Pcon-pT181 attenuator(2-SA)	17.3	1.93	0.03
8/22 spinach-DT (3-2)	22.4	1.93	0.09
8/22 Ptet-PT181 anisense (1-3)	18.3	1.58	0.14
8/22 Pcon-aptamer12_1R-DT (E-1A)	8.2	2.10	0.05
8/22 Pcon-spinach-DT(E-1A)	5.6	0.91	0.05
8/22 Pcon-pT181 attenuator(E-1A)	18.5	1.80	0.12
8/22 DT(1-3)	7.3	1.13	0.04
8/22 tetR aptamer 12_1R-DT(1-3)	14.6	1.74	0.06
8/22 Pcon-pT181 attenuator(1-SA)	7.9	1.57	0.04
8/22 Pcon-RBS-tetR-DT(1-2Ⓐ)	41.6	1.81	0.25
8/22 Pcon-RBS-tetR-DT(3-2Ⓐ)	30.2	1.75	0.15
8/22 Pcon-RBS-tetR-DT(E-2Ⓐ)	36.9	1.75	0.56
8/22 Pcon-RBS-GFP-DT(1-SⒶ)	43.5	1.83	0.33
8/22 Pcon-RBS-GFP-DT(2-SⒶ)	64.6	1.82	0.30
8/23 Pcon-pT181 attenuator-DT (E-1A)	31.2	1.83	0.09
8/23 Pcon-pT181 attenuator-DT (0-1A)	64.5	1.80	0.43
8/23 Pcon-pT181 antisense-spinach-DT (1-2A)	36.4	1.88	0.21
8/23 Pcon-pT181 antisense-spinach-DT (E-1A)	27.9	1.92	0.16
8/23 Ptet(1)(2-S)	37.0	1.91	0.16
9/23 Plac-2(E-1A)	21.2	1.82	0.13

Transformation

No name

Name	Sample	Competent Cells	Total	Plate
9/23 Pcon-attenuator-aptamer12-1R-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 pT181attenuator(1C3)	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181antisense-DT	2 µL	20 µL	22 µL	LB (+CP)
9/23 Pcon-pT181attenuator-RBS--GFP-DT	2 µL	20 µL	22 µL	LB (+CP)

Liquid Culture

No name

Sample	medium
pSB1C3 (Master plate-1)	LB(+CP)

Restriction Enzyme Digestion

Kojima

	8/21 pT181attenuator (330 µg/mL)	XbaI	PstI	buffer	BSA	MilliQ	total
2 cuts	6.1	1	1	3	3	15.9	30
NC	0.3	0	0	1	1	7.7	10

	9/22 Pcon-pT181attenuator (368.3 µg/mL)	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	5.4	1	1	3	0	19.6	30
NC	0.3	0	0	1	0	8.7	10

	9/16 pSB1C3	EcoRI	SpeI	buffer	BSA	MilliQ	total
2 cuts	18	1	1	3	0	7.0	30
NC	0.9	0	0	1	0	8.1	10

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	-	-
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	--	--
4	Pcon-pT181attenuator	EcoRI	SpeI
5	Pcon-pT181attenuator	--	--
6	pSB1C3	EcoRI	SpeI
7	pSB1C3	--	--

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
9/24 aptamer12-1R-DT	144.7	1.03	1.21

Gel Extraction

Kojima

Lane	DNA	Enzyme1	Enzyme2
1	100kb ladder	--	--
2	pT181attenuator	XbaI	PstI
3	pT181attenuator	XbaI	PstI
4	pT181attenuator	XbaI	PstI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	SpeI
7	Pcon-pT181attenuator	EcoRI	SpeI
8	Pcon-pT181attenuator	EcoRI	SpeI
9	--	--	--
10	pSB1C3	EcoRI	SpeI
11	pSB1C3	EcoRI	SpeI
12	pSB1C3	EcoRI	SpeI

Name	concentration[µg/mL]	260/280	260/230
pT181attenuator(XbaI&PstI)	10.1	2.02	0.43
Pcon-pT181attenuator(EcoRI&SpeI)	10.3	1.81	0.32
pSB1C3(EcoRI&SpeI)	10.0	2.81	0.02

Colony PCR

Nakamoto

Sample	base pair
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7	1414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8	1414
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7	1631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8	1631
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3	2042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4	2042
9/23 Pcon-pT181antisense-DT 1	508
9/23 Pcon-pT181antisense-DT 2	508
9/23 Pcon-pT181antisense-DT 3	508
9/23 Pcon-pT181antisense-DT 4	508
9/23 Pcon-pT181antisense-DT 5	508
9/23 Pcon-pT181antisense-DT 6	508
9/23 Pcon-pT181antisense-DT 7	508
9/23 Pcon-pT181antisense-DT 8	508

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
5 min	30 sec	30 sec	2min	30 cycles

Lane	Sample
1	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5
6	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6
7	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7
8	9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8
9	1kb ladder
10	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1
11	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2
12	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3
13	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4
14	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5
15	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6
16	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7
17	9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8

Lane	Sample
1	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1
2	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2
3	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3
4	9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4
5	9/23 Pcon-pT181antisense-DT 1
6	9/23 Pcon-pT181antisense-DT 2
7	1kb ladder
8	9/23 Pcon-pT181antisense-DT 3
9	9/23 Pcon-pT181antisense-DT 4
10	9/23 Pcon-pT181antisense-DT 5
11	9/23 Pcon-pT181antisense-DT 6
12	9/23 Pcon-pT181antisense-DT 7
13	9/23 Pcon-pT181antisense-DT 8
14	--

Restriction Enzyme Digestion

Nakamoto

	9/24 aptamer12-1R-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	17.5µL	1.0µL	1.0µL	3.0µL	3.0µL	4.5µL	30µL
NC	0.9µL	0µL	0µL	1.0µL	1.0µL	7.1µL	10µL

	9/20 Ptet	SpeI	PstI	buffer	BSA	MilliQ	total
2cuts	9.5µL	1.0µL	1.0µL	3.0µL	0µL	15.5µL	30µL
NC	0.5µL	0µL	0µL	1.0µL	0µL	8.5µL	10µL

	9/22 RBS-GFP-DT	EcoRI	SpeI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	0µL	21.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	0µL	8.8µL	10µL

	9/22 Pcon-RBS-GFP-DT	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	2.4µL	1.0µL	1.0µL	3.0µL	3.0µL	19.6µL	30µL
NC	0.1µL	0µL	0µL	1.0µL	1.0µL	7.9µL	10µL

	9/22 Pcon-pT181attenuator 1	EcoRI	XbaI	buffer	BSA	MilliQ	total
2cuts	5.4µL	1.0µL	1.0µL	3.0µL	3.0µL	16.6µL	30µL
NC	0.3µL	0µL	0µL	1.0µL	1.0µL	7.7µL	10µL

	9/22 RBS-GFP-DT	XbaI	PstI	buffer	BSA	MilliQ	total
2cuts	3.8µL	1.0µL	1.0µL	3.0µL	3.0µL	18.2µL	30µL
NC	0.2µL	0µL	0µL	1.0µL	1.0µL	7.8µL	10µL

Ligation

Nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/22 aptamer12-1R-DT(EcoRI&XbaI)	1.4 µL	9/24 Pcon-pT181attenuator(EcoRI&SpeI)	26 µL	3.5 µL
experiment	9/4 Ptet(SpeI&PstI)	2.0 µL	9/21 RBS-GFP-DT(XbaI&PstI)	9.0 µL	3.5 µL

incubated at 37 °C for 1 hour.

colony PCR

Kojima

Sample	base pair
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11	2042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12	2042bp
9/23 Pcon-pT181antisense-DT 9	508bp
9/23 Pcon-pT181antisense-DT 10	508bp
9/23 Pcon-pT181antisense-DT 11	508bp
9/23 Pcon-pT181antisense-DT 12	508bp
9/23 Pcon-pT181antisense-DT 13	508bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 1	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 2	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 3	1527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 4	1527bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3	859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4	859bp
9/24 pT181attenuator(pSB1C3) 1	390bp
9/24 pT181attenuator(pSB1C3) 2	390bp
9/24 pT181attenuator(pSB1C3) 3	390bp
9/24 pT181attenuator(pSB1C3) 4	390bp

Tatsui

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	2min	30cycle

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
pSB1C3	183.9	1.91	1.59
Pcon-pT181attenuator-aptamer12-1R-DT	231.7	1.82	2.35
Plac-aptamer12-1R-DT	208.9	1.98	1.79

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	9/24 aptamer12-1R-DT	EcoRI	XbaI
3	9/24 aptamer12-1R-DT	--	--
4	9/24 Ptet	SpeI	PstI
5	9/24 Ptet	--	--
6	9/24 RBS-GFP-DT	EcoRI	SpeI
7	9/24 RBS-GFP-DT	--	--
8	9/24 Pcon-RBS-GFP-DT	EcoRI	XbaI
8	9/24 Pcon-RBS-GFP-DT	--	--
9	9/24 Pcon-RBS-GFP-DT	--	--
10	9/24 Pcon-pT181attenuator 1	EcoRI	XbaI
11	9/24 Pcon-pT181attenuator 1	--	--
12	9/24 RBS-GFP-DT	XbaI	PstI
13	9/24 RBS-GFP-DT	--	--
14	1kb ladder	--	--

Gel Extraction

No name 1

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	aptamer12-1R-DT	EcoRI	XbaI
3	aptamer12-1R-DT	EcoRI	XbaI
4	aptamer12-1R-DT	EcoRI	XbaI
5	--	--	--
6	Ptet	SpeI	PstI
7	Ptet	SpeI	PstI
8	Ptet	SpeI	PstI
9	--	--	--
10	RBS-GFP-DT	EcoRI	SpeI
11	RBS-GFP-DT	EcoRI	SpeI
12	RBS-GFP-DT	EcoRI	SpeI

Lane	DNA	Enzyme1	Enzyme2
1	1kb ladder	--	--
2	Pcon-RBS-GFP-DT	EcoRI	XbaI
3	Pcon-RBS-GFP-DT	EcoRI	XbaI
4	Pcon-RBS-GFP-DT	EcoRI	XbaI
5	--	--	--
6	Pcon-pT181attenuator	EcoRI	XbaI
7	Pcon-pT181attenuator	EcoRI	XbaI
8	Pcon-pT181attenuator	EcoRI	XbaI
9	--	--	--
10	RBS-GFP-DT	XbaI	PstI
11	RBS-GFP-DT	XbaI	PstI
12	RBS-GFP-DT	XbaI	PstI

Electrophoresis

No name
1

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6
4	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7
5	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8
6	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9
7	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10
8	1kb ladder

2

Lane	Sample
1	1kb ladder
2	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11
3	Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12
4	Pcon-pT181attenuator-RBS-GFP-DT 1
5	Pcon-pT181attenuator-RBS-GFP-DT 2
6	Pcon-pT181attenuator-RBS-GFP-DT 3
7	Pcon-pT181attenuator-RBS-GFP-DT 4
8	1kb ladder

3

Lane	Sample
1	Pcon-pT181antisense-DT 9
2	Pcon-pT181antisense-DT 10
3	Pcon-pT181antisense-DT 11
4	Pcon-pT181antisense-DT 12
5	Pcon-pT181antisense-DT 13
6	100bp ladder
7	Pcon-pT181attenuator-aptamer12-1R-DT 1
8	Pcon-pT181attenuator-aptamer12-1R-DT 2

4

Lane	Sample
1	Pcon-pT181attenuator-aptamer12-1R-DT 3
2	Pcon-pT181attenuator-aptamer12-1R-DT 4
3	100bp ladder
4	pT181attenuator(pSB1C3) 1
5	pT181attenuator(pSB1C3) 2
6	pT181attenuator(pSB1C3) 3
7	pT181attenuator(pSB1C3) 4

Ligation

Hirano

state	Vector		Inserter1		Inserter2		Inserter3		Inserter4		(NEB)T4ligase	(NEB)Buffer	milliQ	total
experiment	RBS-GFP-DT(pSB1C3)	1µL	tetR-aptamer12-1R-DT(1-3)	1 µL	Ptet(2-S)	0.3µL	Pcon-tetR-aptamer12-1R-DT(3-2A)	1.1µL	Plac(E-1A)	0.5µL	0.5µL	0.5µL	0.2 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator(E-1A)	1 µL	Pcon-tetR-aptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0.7 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-tetRaptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0 µL	5.3µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-spinach-DT(E-1A)	3µL	Pcon-tetRaptamer12-1R-DT(1-2A)	1µL	Ptet(2-S)	0.3µL	--	--	0.5µL	0.5µL	0 µL	6.3µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181antisense-DT(E-1A)	0.5µL	DT(1-3)	2µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(3-2A)	1.1µL	0.5µL	0.5µL	0.1 µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator(E-1A)	1µL	tetRaptamer12-1R-DT(1-3)	1µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(3-2A)	1.1µL	0.5µL	0.5µL	0µL	5.4µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Ptet(2-S)	0.3µL	Pcon-tetRaptamer12-1R-DT(E-2A)	1µL	--	--	--	--	0.5µL	0.5µL	1.7µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-GFP-DT(2-SA)	0.5µL	1µL	--	--	--	--	--	0.5µL	0.5µL	1.5µL	5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181attenuator(1-SA)	2.1µL	--	--	--	--	0.5µL	0.5µL	0µL	6.1µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-pT181attenuator-DT(E-1A)	1µL	Pcon-pT181attenuator(1-SA)	2.1µL	--	--	--	--	0.5µL	0.5µL	0µL	5.1µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetR12-1R-DT(E-1A)	2µL	Pcon-GFP-DT(2-SA)	0.5µL	spinach-DT(3-2)	1µL	Ptet-pT181antisense(1-3)	1µL	0.5µL	0.5µL	0µL	6.5µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181antisense(1-2A)	0.7µL	Pcon-pT181attenuator(2-SA)	2.1µL	--	--	0.5µL	0.5µL	0µL	6.8µL
experiment	RBS-GFP-DT(pSB1C3)	1µL	Pcon-tetRaptamer12-1R-DT(E-1A)	2µL	Pcon-pT181attenuator(2-SA)	2.1µL	Spinach-DT(3-2)	1µL	Ptet-pT181antisense(1-3)	1µL	0.5µL	0.5µL	0µL	8.1µL

Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL

PCR

No name

genome DNA	KOD plus	10x buffer	dNTP	MgSO4	F primer	R primer	KOD-plus	MilliQ	total
Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
DT-(1C3-GG1-fwd,1C3-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Ptet(1C3-GGE-fwd,1C3-GG0-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL
DT(1C3-GG2-fwd,1C3-GG1-rev)	1µL	2.5µL	2.5µL	1.5µL	0.75µL	0.75µL	0.5µL	15.5	25µL

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10sec	30sec	36sec	30

Sep 25

Electrophoresis

nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-antisense (1C3-66E-fwd A2-662 rev)	--	--
3	DT (1C3-661-fwd 1C3 662 rev)	--	--
4	spinach-DT(1C3-661-fwd 1C3 662 rev)	--	--
5	Pcon-antisense-spinach-DT(1C3-66E fwd A2-661 rev)	--	--
6	100bp ladder	--	--

Lane	Sample	Enzyme1	Enzyme2
2	100bp ladder	--	--
3	Ptet (1C3 66E fwd-1C3 660 rev)	--	--
4	Pcon-attenuator(1C3-660 fwd A2-661 rev)	--	--
5	DT(1C3 662 fwd-1C3 661 rev)	--	--
6	Pcon-attenuator-aptamer-DT(1C3 66E fwd-A2-661-rev)	--	--
7	100bp ladder	--	--

Colony PCR

Nakamoto

Sample	base pair
9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12)	859
9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8)	1527
9/23 Pcon-PT181 attenuator-pSB1C3-(5~8)	601

PreDenature	Denature	Annealing	Extension	cycle
94 °C	94 °C	55 °C	68 °C	--
2 min	30 s	30 s	1min5s	30 cycles

Electrophoresis

no name

Lane	Sample	Enzyme1	Enzyme2
1	Pcon-PT181 attenuator-aptamer12-1R-DT-5	--	--
2	Pcon-PT181 attenuator-aptamer12-1R-DT-6	--	--
3	Pcon-PT181 attenuator-aptamer12-1R-DT-7	--	--
4	Pcon-PT181 attenuator-aptamer12-1R-DT-8	--	--
5	Pcon-PT181 attenuator-aptamer12-1R-DT-9	--	--
6	Pcon-PT181 attenuator-aptamer12-1R-DT-10	--	--
7	Pcon-PT181 attenuator-aptamer12-1R-DT-11	--	--
8	Pcon-PT181 attenuator-aptamer12-1R-DT-12	--	--
9	1kbp ladder	--	--
10	Pcon-PT181 attenuator-RBS-GFP-DT-5	--	--
11	Pcon-PT181 attenuator-RBS-GFP-DT-6	--	--
12	Pcon-PT181 attenuator-RBS-GFP-DT-7	--	--
13	Pcon-PT181 attenuator-RBS-GFP-DT-8	--	--
14	Pcon-PT181 attenuator-pSB1C3-5	--	--
15	Pcon-PT181 attenuator-pSB1C3-6	--	--
16	Pcon-PT181 attenuator-pSB1C3-7	--	--
17	Pcon-PT181 attenuator-pSB1C3-8	--	--

Transformation

kojima and nakamoto

Name	Sample	Competent Cells	Total	Plate
Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon tetR-DT+Ptet+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT	1µL	10µL	11µL	--

Name	Sample	Competent Cells	Total	Plate
aptamer12-DT(EcoRI+XbaI)+Pcon-pT181attenuator(EcoRI+SpeI)	2µL	20µL	22µL	--
Ptet(SpeI+PstI)+RBS-GFP-DT(XbaI+PstI))	2µL	20µL	22µL	--

Ligation

nakamoto

state	Vector		Inserter		Ligation High ver.2
experiment	9/25 aptamer12_1R-DT(EcoRI&XbaI)	4.5 µL	Pcon-pT181 attenuator(EcoRI&SpeI)	7.7 µL	6.1 µL
experiment	9/17 pSB1C3(EcoRI&SpeI)	2.0 µL	9/3 pT181 attenuator(EcoRI&SpeI)	14 µL	8 µL
experiment	9/25 Pcon-pT181 attenuator(EcoRI&XbaI)	4.5 µL	9/25 RBS-GFP-DT(EcoRI&SpeI)	19.3 µL	11.9 µL
experiment	9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI)	4.2 µL	Pcon-RBS-tetR-DT(EcoRI&SpeI)	20 µL	12.1 µL

Restriction Enzyme Digestion

nakamoto>

	9/24 Pcon-attenuator-aptamer-DT(µL)	EcoRI(µL)	SpeI(µL)	Buffer(µL)	MilliQ(µL)	total(µL)
2 cuts	8.6	1	1	3	16.4	30
NC	0.4	0	0	1	8.6	10

	9/11 DT(µL)	EcoRI(µL)	XbaI(µL)	Buffer(µL)	BSA(µL)	MilliQ(µL)	total(µL)
2 cuts	10.4	1	1	3	3	11.6	30
NC	0.5	0	0	1	1	7.5	10

Liquid Culture

Kojima

Sample	medium
DT	--
pT181attenuator	--
Pcon-RBS-tetR-DT	--

Gel Extraction

Kojima

Lane	DNA	Enzyme
1	100bp ladder	--
2	Pcon-pT181antisense	--
3
4
5	--	--
6	DT	--
7
8
9	--	--
10	Spinach-DT	--
11
12

Lane	DNA	Enzyme
1	100bp ladder	--
3	Pcon-antisense-spinach-DT(E-1A)	--
4	Pcon-antisense-spinach-DT(E-1A)	--
5	Pcon-antisense-spinach-DT(E-1A)	--

Lane	DNA	Enzyme
7	100bp ladder	--
9	Ptet(E-0)	--
10	Ptet(E-0)	--
11	Ptet(E-0)	--

Lane	DNA	Enzyme
13	100bp ladder	--
15	Pcon-attenuator(0-1A)	--
16	Pcon-attenuator(0-1A)	--
17	Pcon-attenuator(0-1A)	--

||--

Lane	DNA	Enzyme
19	100bp ladder	--
21	Pcon-attenuator-aptamer-DT(E-1A)	--
22	Pcon-attenuator-aptamer-DT(E-1A)	--
23	Pcon-attenuator-aptamer-DT(E-1A)	--

Name	concentration[µg/mL]	260/280	260/230
DT(2-1)	24.9	1.73	0.37
Ptet(E-0)	12.8	1.59	0.90
Pcon-antisense(E-2A)	16.5	1.72	1.07
Pcon-attenuator-aptamer-DT(E-1A)	22.1	1.75	0.94
Pcon-antisense-spinach-DT(E-1A)	31.0	1.78	1.15
spinach-DT(1-2)	23.4	1.85	1.11
Pcon-attenuator(0-1A)	22.5	1.76	0.46
DT(1-2)	25.1	1.78	1.21

Electrophoresis

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	Pcon-pT181 attenuator-aptamer 12_1R-DT	EcoRI	SpeI
3	Pcon-pT181 attenuator-aptamer 12_1R-DT	--	--
4	DT	EcoRI	XbaI
5	DT	--	--

Gel Extraction

Nakamoto

Lane	DNA	Enzyme
1	DT	EcoRI+XbaI
2	DT	EcoRI+XbaI
3	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI
4	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI
5	Pcon-pT181 attenuator-aptamer12_1R-DT	EcoRI+SpeI

Name	concentration[µg/mL]	260/280	260/230
Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)	11.8	1.80	0.46
DT(EcoRI+XbaI)	42.6	1.84	0.98

Liquid Culture

No name

Sample	medium
Pcon-pT181anntisense-spinach-DT(4k5)	-

Ligation

Nakamoto and Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	9/24 pSB1C3(EcoRI+Spel)	10µL	9/25 PconpT181attenuator-aptamer12-1R-DT(EcoRI+Spel)	5.9µL	8µL

PCR

Hirano

800pg/μL DT	KOD plus	10x buffer	dNTP	MgSO4	1C3_GG2_fwd	iC3_GG1_rev	MilliQ	total
1	0.5	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	57°C	68°C	--
2min	10s	30s	24s	30cycles

Transformation

Tatsui

Name	Plate
aptamer12-1R-DT	CP
pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal)	CP
Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI)	Amp
Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI)	Amp
pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI)	CP

Electrophoresis

Hirano

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	DT(2-1)	--	--

Column Refining

Name	concentration[µg/mL]	260/280	260/230
8/25 DT(2-1)	--	--	--

Restriction Enzyme Digestion

Concentrate PCR product by evaporator until volume become less than 3µL, add all sulution and pipetting.

9/25 DT(2-1)	10*cut smart Buffer	BsaI-HF	MilliQ	total
3.0 µL	2.0 µL	0.3 µL	14.7 µL	20.0 µL

Incubate 37°C 12hour

Liquid Culture

Hirano

Sample	medium
Pcon-GFP-DT	LB(Amp)
RBS-GFP-DT	LB(CP)
Pcon-spinach-DT	LB(Amp)
Pcon-tetRaptamer-DT	LB(Amp)
Pcon-attenuator-DT	LB(Amp)
spinach-DT	LB(CP)
Pcon-antisense-spinach-DT	LB(Amp)

Plating

Hirano

Mix Liquid Culture 1mL and LB(+Amp), incubate 37°C 30min

OD600-1.445

Sample	Use plate	The number of plate
Pcon-GFP-DT	M9(+Amp)	3
Pcon-GFP-DT	LB(+Amp)	3

</div> incubate 37°C

Colony PCR

No name

Sample	base pair
9/24 GGA-1(1~8)	3177
9/24 GGA-2(1~8)	3189
9/24 GGA-3(1~8)	2989
9/24 GGA-4(1~8)	2939
9/24 GGA-7(1)	2489
GGA-16(1)	2717
RBS-GFP-DT	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	3min15s	30cycles

RNA Extraction

Nakamoto
sumple 0.25mL
Add 1mLISOGEN-LS
Lysis or homogenization
Store for 5 min, at room temperature
Add 0.2mL Chloroform
Shake vigorously for 15sec.
Store for 2~3min.at room temperature
Centrifuge 12K*g for 15min. at 4°C
Extract aqueous phase
Add 0.5mL isopropanol
Store for 5~10min. at room temperature
Centrifuge 12K*g for 10min. at 4°C
Remove aqueous phase
Add at least 1mL 70% ethanol
Vortex
Centrifuge 7.5K*g for 5min. at 4°C
Remove aqueous phase
Dry briefly
Add ddH2O, TE(pH8.0) or 0.5% SDS
Dissolve
Total RNA solution

DNA	concentration[µg/mL]	260/280	260/230
Pcon-RBS-GFP-DT	2783	1.95	1.48

Add 91.2µL, Store at freezer
Total 250µL</div>

cDNA　Synthesis

Nakamoto

volume	7 µL
7*cDNA Wipcout Buffer	1 µL
Template RNA(9/25 Pcon-RBS-GFP-DT)	3 µL
RNase-frec water	4 µL

Incubate 42°C 2min, then on ice immediately
Add

5*Quantiscript Reverse Transcriptase	0.5 µL
5*Quantiscript RT Buffer	2 µL
RT Primer Mix	0.5µL

Incubate 42°C 15min, then incubate 95°C 3min

Electrophoresis

Nakamoto

Lane	Sample	Enzyme1	Enzyme2
1	100bp ladder	--	--
2	Pcon-RBS-GFP-DT(RNA)	--	--

Ligation

Tatsui

state	Vector		Inserter		Ligation High ver.2
experiment	9/13 Pcon-pT181-attenuator(SpeI&PstI)	3.5µL	9/25 RBS-GFP-DT(XbaI&PstI)	13.5µL	8.5 µL

Sep 26

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	GGA1-1	--	--
2	GGA1-2	--	--
3	GGA1-3	--	--
4	GGA1-4	--	--
5	GGA1-5	--	--
6	GGA1-6	--	--
7	GGA1-7	--	--
8	GGA1-8	--	--
9	1kbp ladder	--	--
10	GGA2-1	--	--
11	GGA2-2	--	--
12	GGA2-3	--	--
13	GGA2-4	--	--
14	GGA2-5	--	--
15	GGA2-6	--	--
16	GGA2-7	--	--
17	GGA2-8	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA3-1	--	--
2	GGA3-2	--	--
3	GGA3-3	--	--
4	1kbp ladder	--	--
5	GGA3-4	--	--
6	GGA3-5	--	--
7	GGA3-6	--	--
8	GGA3-7	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA3-8	--	--
2	GGA4-1	--	--
3	GGA4-2	--	--
4	GGA4-3	--	--
5	1kbp ladder	--	--
6	GGA4-4	--	--
7	GGA4-5	--	--
8	GGA4-6	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA4-7	--	--
2	GGA4-8	--	--
3	1kbp ladder	--	--
4	GGA7-1	--	--
5	GGA16-1	--	--
6	NC(RBS-GFP-DT)	--	--

Ligasion(Golden Gate Assenbly)

No name

state	Vector		Inserter1		Inserter2		Inserter3		Inserter4		Inserter5		(NEB)T4 ligase	(NEB)10*T4 ligase	MilliQ
experiment	RBS-GFP-DT	1.0 µL	Pcon-pT181 antisense(E-2A)	0.6µL	DT(2-1)	0.5µL	Pcon-pT181 attenuator(1-SA)	2.1 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-pT181 antisense(E-2A)	0.9 µL	DT(2-1)	0.5 µL	Pcon-GFP-DT(1-SA)	1.5 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Ptet(E-0)	0.7µL	Pcon pT181 attenuator(0-1A)	0.3µL	tet aptamer(1-3)	0.3 µL	Pcon-tetR-DT(3-2A)	1.1 µL	Ptet(2-5)	0.3 µL	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Ptet(E-0)	0.7 µL	Pcon-pT181 attenuator-DT(0-1A)	1.0 µL	Pcon-tetR-DT(1-2A)	1.0 µL	Ptet(2-5)	0.3µL	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Pcon-pT181 attenuator-tetR-DT(E-1A)	1.0 µL	Pcon antisense-spinach-DT(1-2A)	0.7µL	Pcon-pT 181 attenuator(2-SA)	2.1 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	RBS-GFP-DT	1.0 µL	Pcon antisense(E-1A)	0.5 µL	spinach-DT(1-2)	1.0 µL	Pcon-GFP-DT(1-2)	0.8 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-pT181 antisense(E-1A)	0.8µL	spinach-DT(1-2)	1.0µL	Pcon-GFP-DT(2-SA)	0.8 µL	--	--	--	--	0.5 µL	0.5 µL	up to 5 µL
experiment	pSB1C3(EcoRI&SpeI)	1.8 µL	Pcon-tet aptamer-DT(E-1A)	3.0 µL	Ptet-pT181 antisense(1-3)	1.5 µL	spinach-DT(3-2)	1.5 µL	Pcon-GFP-DT(2-5A)	0.8 µL	--	--	0.5 µL	0.5 µL	up to 5 µL

Liquid Culture

No name

Sample	medium
Pcon-GFP-DT	Amp
RBS-GFP-DT	CP
Pcon-pT181 attenuator-DT	Amp
Pcon-tet aptamer-DT	Amp
Pcon-spinach-DT	Amp
Pcon-pT181 antisense-spinach-DT	Amp
spinach-DT	CP

BSAI Digestion

No name

	DNA	MilliQ	BSAI-HF	10*cut smart	total
Pcon	7 µL	10.7 µL	0.3 µL	2 µL	20 µL
RBS-luxR-DT	10 µL	7 µL	0.3 µL	2 µL	20 µL

Colony PCR

Tatsui

Sample	base pair
GGA-1 (9~16)	3177
GGA-2 (9~16)	3189
GGA-3 (9~21)	2989
GGA-4 (9~16)	2939
GGA-6 (1)	3359
GGA-16(2~4)	2939

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	3min30s	30cycles

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	GGA-1 (9)	--	--
2	GGA-1 (10)	--	--
3	GGA-1 (11)	--	--
4	GGA-1 (12)	--	--
5	GGA-1 (13)	--	--
6	GGA-1 (14)	--	--
7	GGA-1 (15)	--	--
8	GGA-1 (16)	--	--
9	1kbp ladder	--	--
10	GGA-2 (9)	--	--
11	GGA-2 (10)	--	--
12	GGA-2 (11)	--	--
13	GGA-2 (12)	--	--
14	GGA-2 (13)	--	--
15	GGA-2 (14)	--	--
16	GGA-2 (15)	--	--
17	GGA-2 (16)	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA-4 (9)	--	--
2	GGA-4 (10)	--	--
3	GGA-4 (11)	--	--
4	GGA-4 (12)	--	--
5	GGA-4 (13)	--	--
6	GGA-4 (14)	--	--
7	GGA-4 (15)	--	--
8	GGA-4 (16)	--	--
9	1kbp ladder	--	--
10	GGA-6 (1)	--	--
11	GGA-16 (2)	--	--
12	GGA-16 (3)	--	--
13	GGA-16 (4)	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA-3 (9)	--	--
2	GGA-3 (10)	--	--
3	GGA-3 (11)	--	--
4	GGA-3 (12)	--	--
5	1kbp ladder	--	--
6	GGA-3 (13)	--	--
7	GGA-3 (15)	--	--
8	GGA-3 (14)	--	--

Lane	Sample	Enzyme1	Enzyme2
1	GGA-3 (17)	--	--
2	GGA-3 (18)	--	--
3	GGA-3 (19)	--	--
4	1kbp ladder	--	--
5	GGA-3 (20)	--	--
6	GGA-3 (21)	--	--
7	GGA-3 (16)	--	--

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT(RNA)	--	--
2	1kbp ladder	--	--
3	Pcon-RBS-GFP-DT(RNA)	--	--

Liquid Culture

Hirano

Sample	medium
Pcon-GFP-DT	--
RBS-GFP-DT	--
Pcon-tet aptamer-DT	--
Pcon-spinach-DT	--
Pcon-pT181 antisense-spinach-DT	--
spinach-DT	--

Colony PCR

Hirano

Sample	base pair
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-1	--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-2	--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-3	--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-4	--
9/24 Ptet-RBS-GFP-DT-1	--

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min40s	30cycles

Ligasion(Golden Gate Assenbly)

Hirano

state	Vector	Inserter1	Inserter2	Inserter3	Inserter4	Inserter5
experiment	GGA2	Pcon-attenuator(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA3	Pcon-tetap-DT(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA4	Pcon-spi-DT(E-1A)	Pcon-tetR-DT(1-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA5	Pcon-antisense(E-1A)	DT(1-3)	Pcon-tetR-DT(3-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA6	Pcon-attenuator(E-1A)	tetap-DT(1-3)	Pcon-tetR-DT(3-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)
experiment	GGA7	Pcon-tetR-DT(E-2A)	Ptet(2-S)	RBS-GFP-DT(EcoRI&XbaI)

PreDenature	Denature	Annealing	Extension	cycle
37°C	16°C	50°C	80°C	--
3min	4min	5min	5min	25cycles

RNA Extraction

Nakamoto

Sample
Pcon-Spinach-DT
Pcon-tetRaptamer-DT
Pcon-attenuator-DT
Spinach-DT
Pcon-antisense-Spinach-DT

Liquid Culture

Kozima

Sample
Pcon-attemuater-DT
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-RBS-tetR-DT
Pcon-antisense-spinach-DT(Master 1)
Pcon-antisense

Colony PCR

Nakamoto Kozima

Sample	base pair
9/25 9/19pSB1C3&9/3PT181 attenuator(1~3)	601bp
9/25 aptamer12_1R-DT&9/24Pcon-pT181 attenuator(1~4)	859bp
Pcon-attenuator-aptamer-DT(1)	859bp

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	65°C	--
5min	30sec	30sec	54sec	30

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	1kbp ladder	--	--
2	9/25 9/23 1C3&9/3attenuator	--	--
3	9/25 9/23 1C3&9/3attenuator	--	--
4	9/25 9/23 1C3&9/3attenuator	--	--
5	9/25aptamer-DT&9/24Pcon-attenuator	--	--
6	9/25aptamer-DT&9/24Pcon-attenuator	--	--
7	9/25aptamer-DT&9/24Pcon-attenuator	--	--
8	9/24 Pcon attenuator-aptamer-DT(1)	--	--
9	9/25aptamer-DT&9/24Pcon-attenuator	--	--

Plating

Hirano

Sample	Use plate
9/26 GGA2	LB(CP)
9/26 GGA3	LB(CP)
9/26 GGA4	LB(CP)
9/26 GGA5	LB(CP)
9/26 GGA6	LB(CP)
9/26 GGA7	LB(CP)
9/26 GGA11	LB(CP)
9/26 GGA312	LB(CP)
9/26 GGA13	LB(CP)
9/26 GGA14	LB(CP)
9/26 GGA15	LB(CP)
9/26 GGA20	LB(CP)
9/26 GGA23	LB(CP)
9/26 GGA24	LB(CP)
9/26 Ptet+RBS-GFP-DT	LB(CP)

incubate 37°C

Restriction Enzyme Digestion

Kojima

	9/24 pSB1C3	EcoRI	SpeI	buffer	MilliQ	total
2cuts	11µL	1µL	1µL	3µL	14µL	30µL
NC	0.5µL	0µL	0µL	1µL	8.5µL	10µL

	9/24 Pcon-pT181attenuator-aptamer12-1R-DT	EcoRI	SpeI	buffer	MilliQ	total
2cuts	8.7µL	1µL	1µL	3µL	16.3µL	30µL
NC	0.4µL	0µL	0µL	1µL	8.6µL	10µL

	9/24 pT181attenuator	EcoRI	SpeI	buffer	MilliQ	total
2cuts	3.1µL	1µL	1µL	3µL	21.9µL	30µL
NC	0.2µL	0µL	0µL	1µL	8.8µL	10µL

	8/17 RBS-GFP-DT	EcoRI	BSA	buffer	MilliQ	total
1cut	16.4µL	1µL	3µL	3µL	6.6µL	30µL
NC	0.4µL	0µL	1µL	1µL	7.6µL	10µL

	9/17 pSB4K5	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	8.5µL	1µL	1µL	3µL	3µL	13.5µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/14 pSB4K5	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	7.3µL	1µL	1µL	3µL	3µL	14.7µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/22 Pcon-pT181attenuator-DT	EcoRI	SpeI	buffer	MilliQ	total
2cuts	5.4µL	1µL	1µL	3µL	19.6µL	30µL
NC	0.3µL	0µL	0µL	1µL	8.7µL	10µL

	8/7 RBS-GFP-DT 1	XbaI	PstI	BSA	buffer	MilliQ	total
2cuts	8.1µL	1µL	1µL	3µL	3µL	13.9µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

	9/26 RBS-GFP-DT 1	XbaI	BSA	buffer	MilliQ	total
1cut	8.1µL	1µL	3µL	3µL	14.9µL	30µL

	9/16 Pλ-luxI	EcoRI	PstI	BSA	buffer	MilliQ	total
2cuts	14.7µL	1µL	1µL	3µL	3µL	7.3µL	30µL
NC	0.7µL	0µL	0µL	1µL	1µL	7.3µL	10µL

	9/21 Ptet	EcoRI	SpeI	buffer	MilliQ	total
2cuts	14.2µL	1µL	1µL	3µL	10.8µL	30µL
NC	0.7µL	0µL	0µL	1µL	8.3µL	10µL

	9/24 Pcon-pT181attenuator-aptamer12-1R-DT	XbaI	PstI	BSA	buffer	MilliQ	total
2cuts	8.7µL	1µL	1µL	3µL	3µL	13.3µL	30µL
NC	0.4µL	0µL	0µL	1µL	1µL	7.6µL	10µL

Electrophoresis

Kojima

Lane	Sample	Enzyme1	Enzyme2
1	pSB1C3	EcoRI	SpeI
2	pSB1C3	--	--
3	Pcon-pT181attenuator-aptamer12-1R-DT	EcoRI	SpeI
4	Pcon-pT181attenuator-aptamer12-1R-DT	--	--
5	1kb ladder	--	--
6	pT181attenuator	EcoRI	SpeI
7	pT181attenuator	--	--
8	RBS-GFP-DT	EcoRI	--
9	RBS-GFP-DT	--	--

Sep 27

Gel Extraction

Kojima

Lane	DNA	Enzyme
1	1kbp ladder	--
2	pSB1C3	EcoRI+SpeI
3
4
6	Pcon-attenuator-aptmer-DT	EcoRI+SpeI
7
8
10	Pcon-attenuator-aptmer-DT	EcoRI+SpeI
11
12

Name	concentration[µg/mL]	260/280	260/230
Pcon-attenuator-DT(EcoRI+SpeI)	6.8	1.98	0.43
pT181-attenuator(EcoRI+SpeI)	7.5	1.84	0.06
pSB1C3(EcoRI+SpeI)	17.7	1.97	0.71

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	pSB4K5	EcoRI+PstI
4	pSB4K5	EcoRI+PstI
5	pSB4K5	EcoRI+PstI
7	pSB6A1	EcoRI+PstI
8	pSB6A1	EcoRI+PstI
9	pSB6A1	EcoRI+PstI

Electrophoresis

No name

Lane	Sample	Enzyme1	Enzyme2
1	pSB4K5	EcoRI	PstI
2	pSB4K5	-	-
3	1kbp ladder	-	-
4	Pcon-attenuator	EcoRI	SpeI
5	Pcon-attenuator	-	-

Lane	Sample	Enzyme1	Enzyme2
1	RBS-GFP-DT	XbaI	PstI
2	RBS-GFP-DT	-	-
3	RBS-GFP-DT	EcoRI	XbaI
4	1kbp ladder	-	-
5	Ptet	EcoRI	SpeI
6	Ptet	-	-

Liquid Culture

No name

Sample	medium
Pcon-GFP	Amp
RBS-GFP-DT	CP
Pcon-attenuator	Amp
Pcon-attenuator-aptamer-DT	Amp
Pcon-tetR-DT	Amp

Ligation

No name

state	Vector		Inserter		Ligation High ver.2
experiment	9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL	10µL	9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL	1.3µL	3.5 µL
experiment	9/25 DT (EcoRI+XbaI) 42.8ng/µL	2.3µL	9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL	1.5µL	1.9 µL
experiment	9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL	7.8µL	9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL	8.0µL	3.5 µL
experiment	9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL	7.8µL	9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL	12µL	3.5 µL
experiment	9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µL	5.7 µL	8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL	12.6µL	3.5 µL

Liquid Culture

Hirano

Sample	medium
Pcon-spinach-DT	plusgrow Amp
Pcon-tetRaptamer-DT	plusgrow Amp
Pcon-antisense-spinach-DT	plusgrow Amp
spinach-DT	plusgrow CP

RNA Extraction

Nakamoto

Name	concentration[µg/mL]	260/280	260/230
9/26 Pcon-GFP-DT	423.0	1.83	1.46
9/26 Pcon-GFP-DT K	298.7	1.82	1.50

Electrophoresis

No name

Lane	Sample
1	9/27 Pcon-GFP-DT(RNA)
2	1kb ladder
3	9/27 Pcon-GFP-DT K(RNA)

Miniprep

Tatsui

DNA
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-antisense
Pcon-tetR-DT
Pcon-attenuator-DT
Pcon-antisense-spinach-DT

Transformation

Hirano

Name1	Name2	Sample1(µL)	Sample2(µL)	Competent Cells(µL)	Total(µL)
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-attenuator-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-antisense-spinach-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	Pcon-tetR aptamer-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-attenuator-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-antisense-spinach-DT(1A2)	2	1	30	33
Pcon-RBS-GFP-DT(4K5)	Pcon-tetR aptamer-DT(1A2)	2	1	30	33
Pcon-attenuator+RBS-GFP-DT(4K5)	--	2	--	20	22
Pcon-RBS-GFP-DT(4K5)	--	2	--	20	22
attenuator(1C3)	--	2	--	20	22

Miniprep

No name

DNA	concentration[µg/mL]	260/280	260/230
Pcon-RBS－GFP-DT	137.6	1.95	1.78
RBS-GFP-DT	77.0	2.06	1.96
Pcon-PT181 attenuator	231.0	1.89	1.89
Pcon-antisense-Spinach-DT(2)	130.0	1.56	1.62
Pcon-aptamer-DT	218.8	1.92	1.72
Pcon-attenuator-DT	180.7	1.95	1.78
Pcon-Spinach-DT	177.6	1.96	1.06
Pcon-antisense-Spinach-DT(1)	181.1	1.96	1.92
Pcon-antisense	152.9	2.02	1.96
Pcon-RBS+ezR-DT	164.1	1.93	1.91

Restriction Enzyme Digestion

No name

	9/27 RBS-GFP-DT	XbaI	PstI	Buffer	BSA	MilliQ	total
2 cuts	12µL	1µL	1µL	3µL	3µL	20µL	40µL
NC	1.3µL	0µL	0µL	1µL	1µL	6.7µL	10µL

	9/27 Pcon-tetR-DT	EcoRI	PstI	Buffer	MilliQ	total
2 cuts	12.2µL	1µL	1µL	3µL	12.8µL	30µL
NC	0.6µL	0µL	0µL	1µL	8.4µL	10µL

RNA Extraction

Nakamoto

Name	concentration[µg/mL]	260/280	260/230
9/27 Pcon-RBS-GFP-DT	686.6	1.99	1.34
9/27 Pcon-RBS-GFP-DT (+High salt)	127.6	1.76	0.27

DNA sythesis

No name

genome DNA	DNA volume	genome DNA wipeout buffer	RNase freewater	RT	RT buffer	Primer Mix	total
9/27 Pcon-RBS-GFP-DT	0.8µL	1µL	5.2µL	0.5µL	2µL	0.5µL	10µL
9/27 pcon-RBS-GFP-DT(+high salt)	2.5µL	1µL	3.5µL	0.5µL	2µL	0.5µL	10µL
9/27 Pcon-RBS-GFP-DT(revised concentration)	0.5µL	1µL	5.5µL	0.5µL	2µL	0.5µL	10µL

Electrophoresis

No name

Lane	Sample
1	1kb ladder
2	Pcon-RBS-GFP-DT
3	Pcon-RBS-GFP-DT

Liquid Culture

No name

Sample	medium
Pcon spinach DT	plusgrow 2µL(Amp)
Pcon aptamer DT	plusgrow 2µL(Amp)
Pcon antisense Spinach DT	plusgrow 2µL(Amp)
Spinach DT	plusgrow 4µL(CP)

RT PCR

No name

genome DNA	KOD plus	10x buffer for KOD-plus-ver.2	2nM dNTPs	25mM MgSO4	F primer	R primer	MilliQ	total
0.5	1	2.5	2.5	1.5	0.75	0.75	15.5	25

PreDenature	Denature	Annealing	Extension	cycle
94°C	98°C	49.8°C	68°C	--
2min	10sec	30sec	10sec	30

Electrophoresis

No name

Lane	Sample
1	Pcon-GFP-DT(GFP)
2	Pcon-GFP-DT(internal)
3	Pcon-GFP-DT(HS)(GFP)
4	Pcon-GFP-DT(internal)

Lane	Sample
1	Pcon-GFP-DT(GFP)
2	Pcon-GFP-DT(internal)
3	100bp ladder

Digesting cut check

No name

Lane	Sample
1	RBS-GFP-DT(XbaI&PstI)
2	RBS-GFP-DT NC(XbaI&PstI)
3	1kbp ladder
4	Pcon-RBS-tetR-DT(EcoRI&SpeI)
5	Pcon-RBS-tetR-DT NC(EcoRI&SpeI)

Gel Extraction

No name

Lane	DNA	Enzyme
1	1kbp ladder	--
3	RBS-GFP-DT	Xbal+PstI
4	RBS-GFP-DT	Xbal+PstI
5	RBS-GFP-DT	Xbal+PstI
7	1kbp ladder	--
9	Pcon-tetR-DT	EcoRI+SpeI
10	Pcon-tetR-DT	EcoRI+SpeI
11	Pcon-tetR-DT	EcoRI+SpeI

RNA Extraction

Nakamoto

	0	5min	15min	20min	23min	28min	33min	36min
aptamer generator	0.348	0.408	0.550	0.865
Pcon-spinach-DT	0.250	0.240	0.305	0.426	0.471	0.554	0.668	0
GFP generator	0.389	0.420	0.516	0.884
antisense-spinach	0.371	0.438	0.537	0.733
Spinach-DT	0.084	0.110	0.134	0.433	0.269	0.326	0.409	0.742

Name	concentration[µg/mL]	260/280	260/230
Spinach-DT	83.9	1.63	0.17
Pcon-tetRaptamer-DT	425.1	2.10	0.64
Pcon-RBS-GFP-DT	262.4	2.01	0.84
Pcon-antisense-Spinach-DT	489.1	2.05	0.68
Pcon-Spinach-DT	266.6	2.01	0.66

cDNA Synthesis

Nakamoto

	9/27 Spinach-DT	9/23 Pcon-tetRaptamer-DT	9/23 Pcon-RBS-GFP-DT	9/23 Pcon-antisense-spinach-DT	9/23 Pcon-Spinach-DT
RNA	1	1	1	1	1
gDNA wipeout	6	1.2	1.9	1.0	1.9
RNase-free water	0	4.8	4.1	5.0	4.1
RT	0.5	0.5	0.5	0.5	0.5
RT Buffer	2	2	2	2	2
Primer mix	0.5	0.5	0.5	0.5	0.5
total	10µL	10µL	10µL	10µL	10µL

Sep 28

Electrophoresis

Tatsui

Lane	Sample
1	100bp ladder
2	Spinach-DT+GAPDH+A1
3	Spinach-DT+Spinach+C1
4	Spinach-DT+gfp+E1
5	Spinach-DT+aptamer+G1
6	Pcon-tetR aptamer-DT+GADPH+A3
7	Pcon-tetR aptamer-DT+Spinach+C3
8	Pcon-tetR aptamer-DT+gfp+E3
9	Pcon-tetR aptamer-DT+aptamer+G3
10	Pcon-RBS-GFP-DT+GADPH+A5
11	Pcon-RBS-GFP-DT+Spinach+C5
12	Pcon-RBS-GFP-DT+gfp+E5
13	Pcon-RBS-GFP-DT+aptamer+G5
14	100bp ladder

File:Igku xxxxxx.xxx

Lane	Sample
1	Pcon-antisense-Spinach-DT+GAPDH+A7
2	Pcon-antisense-Spinach-DT+Spinach+C7
3	Pcon-antisense-Spinach-DT+gfp+E7
4	Pcon-antisense-Spinach-DT+aptamer+G7
5	100bp ladder
6	Pcon-Spinach-DT+GAPDH+A9
7	Pcon-Spinach-DT+Spinach+C9
8	Pcon-Spinach-DT+gfp+E9
9	Pcon-Spinach-DT+aptamer+G9

File:Igku xxxxxx.xxx

Colony PCR

Sample	base pair
9/27 pT181 attenuatorC(C3)1	330
9/27 pT181 attenuatorC(C3)2	330
9/27 Pcon antisense-DT1	372
9/27 Pcon antisense-DT2	372

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	24s

Sample	base pair
9/27 Pcon-RBS-GFP-DT (4&5)	1158
9/27 Pcon-RBS-GFP-DT (4&5)	1158
9/27 Pcon-RBS-GFP-DT (4&5)	1158
9/27 Pcon-RBS-GFP-DT (4&5)	1158

PreDenature	Denature	Annealing	Extension	cycle
94°C	94°C	55°C	68°C	--
5min	30s	30s	1min24s

Electrophoresis

No name

Lane	Sample
1	100bp ladder
2	Pcon-tetR aptamer-DT+GADPH+A3
3	Pcon-antisense-Spinach-DT+GADPH+A7
4	Spinach-DT+GADPH+A1
5	Pcon-RBS-GFP-DT+GADPH+A5
6	Pcon-Spinach-DT+GADPH+A9

Lane	Sample
1	100bp ladder
2	Pcon-tetR aptamer-DT+gfp+E3
3	Pcon-antisense-Spinach-DT+gfp+E7
4	Spinach-DT+gfp+E1
5	Pcon-RBS-GFP-DT+gfp+E5
6	Pcon-Spinach-DT+gfp+E9

Lane	Sample
1	100bp ladder
2	Pcon-tetR aptamer-DT+Spinach+C3
3	Pcon-antisense-Spinach-DT+gfp+C7
4	Spinach-DT+Spinach+C1
5	Pcon-RBS-GFP-DT+Spinach+C5
6	Pcon-Spinach-DT+Spinach+C9

Lane	Sample
1	100bp ladder
2	Pcon-tetR aptamer-DT+aptamer+G3
3	Pcon-antisense-Spinach-DT+aptamer+G7
4	Spinach-DT+aptamer+G1
5	Pcon-RBS-GFP-DT+aptamer+G5
6	Pcon-Spinach-DT+aptamer+G9

@@ Line 59: / Line 59: @@
 </div>
+{{Kyoto/import|pagename={{PAGENAME}}}}
 {{Kyoto/footer}}

Team:Kyoto/Notebook

From 2013.igem.org

Revision as of 02:03, 28 September 2013

Contents

Calender

Aug 8

Transformation

Aug 9

Transformation

Aug 10

Colony PCR

Electrophoresis

Liquid Culture

LB Medium Plate(+Ampicillin)

Transformation

Aug 11

Miniprep

Colony PCR

Restriction Enzyme Digestion

Electrophoresis

Aug 15

Liquid Culture

Transformation

Miniprep

Aug 16

Colony PCR

Electrophoresis

Master Plate

Transformation

Liquid Culture

Aug 17

Colony PCR

Electrophoresis

Liquid Culture

Master Plate

Miniprep

Restriction Enzyme Digestion

Electrophoresis

Miniprep

Aug 18

Gel Extraction

Ligation

Restriction Enzyme Digestion

Electrophoresis

Colony PCR

Liquid Culture

LB Medium Plate

Transformation

Electrophoresis

Aug 19

Colony PCR

Gel Extraction

LB Medium Plate

Restriction Enzyme Digestion

Ligation

Electrophoresis

Transformation

Gel Extraction

Liquid Culture

Master Plate

Aug 20

Miniprep

LB Medium Plate

Ristriction Enzyme Digestion

Transformation

Electrophoresis

Aug 21

Plusgrow Medium

Liquid culture

Gel Extraction

Ristriction Enzyme Digestion

Electrophoresis

Gel Extraction

Electrophoresis

Ligation

Transformation

LB Medium Plate

Gel Extraction

Miniprep

Aug 22