Team:Kyoto/Notebook2

Notebook2

Measurement of R0011

Wednesday, August 11 By: Wataru

Overnight culture

Name	Strain	Medium	Inducer(IPTG)	Incubation
ML-	KRX	LB (Kan+), 3mL	0mM	At 37℃ 20:20- (Overnight)
ML+	KRX		1mM
MS-	KRX		0mM
MS+	KRX		1mM
KRX	KRX	SOC, 3mL	0mM

Thursday, August 12 By: Tomonori, Takuya O.

Culture

We stopped overnight cultures at 11:10 and took out 30µL culture from each test tube, and added it to 3mL fresh medium which is same for overnight culture. They are grown at 37℃ from 11:10-14:10. After 3-hours culture, OD600 of each culture were measured. Because we found that the 3-hours culture of the sample KRX had not grown, we use the overnight culture of the sample KRX in later procedures. We took out 1mL medium from test tubes and put them into 1.5mL tubes and freeze them. The data is shown in table below.

Measure

We melt samples and centrifuge them at 14,000rpm, at 4℃, for 2min. Discard the supernatant and 150µL cel lysis reagent to each pellet. These pellets were dissolved by vortex for 2min and centrifuged for 2min. From each tubes, 100µL of the supernatant was took out and added to each well of the plate for the plate reader. 100µL cell lysis reagent was also added to them. We measured GFP fluorescence in each sample (Ex/Em = 485/535nm, 1sec). The data is shown in table below. We calculated the promoter activity and the results are shown in table below.

Name	Optical Density (600nm)	Fluorescence (485/535nm)	Calculation 1	Calculation 2	Calculation 3	Promoter Activity
ML-	0.531	4881	4697	8.85 × 103	6.40 × 103	8.65 × 10-2
ML+	0.546	91549	91365	1.67 × 105	1.65 × 105	2.04
MS-	0.645	49469	49285	7.64 × 104	7.40 × 104	1 (0mM IPTG)
MS+	0.623	52017	51833	8.32 × 104	8.08 × 104	1 (1mM IPTG)
KRX	1.413	3644	3460	2.449 × 103	-	-
Cell Lysis Reagent	-	184	-	-	-	-

• Calculation 1: Fluorescence - Fluorescence_{Cell Lysis Reagent}
• Calculation 2: Calculation 1 / Optical Density (600nm)
• Calculation 3: Calculation 2 - Calculation 2_KRX
• Promoter Activity: Calculation 3_ML- / Calculation 3_MS-, Calculation 3_ML+ / Calculation 3_MS+

Friday, August 20 By: Tomonori

Make M9 Medium

M9 salts were added to 1l MilliQ and sterilized by autocrave. M9 medium was stored in a fridge.

Monday, August 23 By: Tasuku, Takuya O.

Make Supplemented M9 Medium

We tried to dissolve CaCl₂ 0.1M (final concentration) in 1l M9 medium, but we failed because CaCl₂ was too much.

Tuesday, August 24 By: Tasuku, Takuya O.

Make Supplemented M9 Medium

M9 salts were added to 1l MilliQ and sterilized by autocrave. We changed amount of CaCl₂ from 0.1M (final concentration) to 0.01mM (final concentration), and we could make supplemented M9 medium.

Overnight culture

The ML, i.e., E.coli KRX transformed with <partinfo>K358000</partinfo> was grown in the supplemented M9 medium including 1mM IPTG for a night (16h).

Wednesday, August 25 By: Tasuku, Takuya O.

Culture

OD600 of the overnight culture was measured. The overnight culture was diluted in fresh supplemented M9 medium including 7 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.

	0.00mM	0.01	0.03	0.05	0.07	0.10	1.00
0h	-0.003	0.014	0.000	-0.008	-	-	-
4h	0.001	0.100	0.033	0.062	-	-	-
6h	-	-	-	-	0.118	0.187	0.178
8h	0.227	0.402	0.363	0.346	0.360	0.479	0.517
16	1.117	1.142	1.131	1.237	1.211	1.202	1.156
20h	1.107	1.118	1.084	1.178	1.177	1.178	1.131

• The OD600 of medium including 0.07mM, 0.10mM, and 1mM IPTG were not measured at 4h, but at 6h.
• Measurement at 16h and 20h was conducted the day after.

We found that the number of the cell was in steady state at 16h and after.

Overnight Culture

The MS, i.e., E.coli KRX transformed with <partinfo>I20260</partinfo> was grown in supplemented M9 medium including 1mM IPTG for a night for 16 hours.

Thursday, August 26 By: Tasuku, Takuya O.

Culture

OD600 of the overnight culture was measured. The overnight culture was diluted in fresh supplemented M9 medium including 7 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.

Time	0.00mM	0.01	0.03	0.05	0.07	0.10	1.00
0h	-0.016	-0.015	0.000	-0.016	-0.018	0.001	-0.007
4	0.108	0.112	0.116	0.094	0.127	0.116	0.160
8	0.626	0.585	0.591	0.522	0.516	0.562	0.553
16	1.351	1.425	1.442	1.483	1.346	1.420	1.497
20	1.363	1.405	1.403	1.442	1.383	1.361	1.434
24	-	1.297	-	-	-	-	1.352

• Measurement at 16h, 20h, and 24h was conducted the day after.

Measure OD600 of the culture of ML and collect samples

Friday, August 27 By: Tasuku, Takuya O.

Measure OD600 of the culture of ML and collect samples

Make the plate of E.coli C2

The E.coli competent cell, C2, was diluted to three different concentration, that is 10^-3, 10^-5, 10<sup-6</sup>. The 200µL of each culture was plated without antibiotics.

Monday, August 30 By: Tomonori, Tasuku

Overnight culture

A colony of each plate made on Friday was picked out and put into the test tube including 5mL supplemented M9 medium without antibiotics. They were grown at 37℃ for a night (16 hours).

Tuesday, August 31 By: Tomonori, Tasuku, Kazuya

Measure OD600 of the overnight culture

OD600 of the overnight culture was measured. We found that E.coli C2 had not grown.

Name	OD600
C2 (diluted to 10-3)	0.034
C3 (diluted to 10-5)	0.062
C4 (diluted to 10-6)	0.040

Because we noticed that the color of the three overnight cultures were strange, we suspected that other bacterium had grwon in these culture.

Culture E.coli C2 wighout antibiotics again

A colony of paltes were picked out and put into the test tube including 5mL supplemented M9 medium wighout antibiotics. They were grown at 37℃ for 11 hours. OD600 of the culture was measured. As the previous experiment, C2 did not grow.

Name	OD600
C2 (diluted to 10-3)	0.031
C3 (diluted to 10-5)	0.042
C4 (diluted to 10-6)	0.091

Overnight culture E.coli C2 in new medium

The supllemented M9 medium wighout antibiotics was made again. E.coli C2 (diluted to 10^-3) was grown for a night (16 hours).

Measure the fluorescence of GFP of the ML and the MS

The fluorescence of GFP of the ML and the MS was measured (Ex/Em = 485/535 nm, 1sec).

ML

Time	0.00mM	0.01	0.03	0.05	0.07	0.10	1.00
0h	51	329	57	62	68	40	5
4	1261	6121	3046	4317	2606	13677	12120
8	36181	74292	67032	65566	42850	78761	68736
16	5573	7274	4759	3526	6131	2844	3091
20	1229	2990	1512	3189	3579	7919	2437

• Back corrected.

MS

Time	0.00mM	0.01	0.03	0.05	0.07	0.10	1.00
0h	3	-13	-11	16	56	32	-14
4	17638	7790	13413	8056	19632	8090	8800
8	66508	85100	236319	83459	126515	177323	84395
16	6504	7556	6159	7650	4228	4240	10662
20	7835	11638	9070	15135	15383	8681	18276
24	-	22526	-	-	-	-	34230

• Back corrected.

Because amounts of samples collected at 0h are very little and the data indicates that is not enough to measure OD600 and fluorescence of GFP correctoly, we decide to measure OD600 after 4 hours only. We calculated the promoter activity of the lactose promoter.

Time	0.00mM	0.01	0.03	0.05	0.07	0.10	1.00
0h	-	-	-	-	-	-	-
4	0.000000	0.880041	0.798267	0.812454	0.142867	1.048718	1.237998
8	1.500221	1.270405	0.461811	1.185223	0.485463	0.521131	0.871168
16	1.036360	1.201241	0.985163	0.552576	1.611748	0.792406	0.375426
20	0.193135	0.322870	0.215761	0.257924	0.273380	1.053934	0.169068

• The autofluorescence of E.coli KRX is not corrected.

Though we drew the graph, we couldn't find any tendency. We decide to conduct experiment with more samples and measure the autofluorescence of E.coli.

Wednesday, September 1 By: Tomonori, Tasuku

Measure OD600 of the overnight culture of C2

OD600 of the overnight culture of C2 was measured. In this experiment, C2 grew.

Name	OD600
C2 (diluted to 10-3)	1.172
C3 (diluted to 10-5)	1.774
C4 (diluted to 10-6)	1.765

We decide not to culture of C2 for measurement of promoter activity because other bacterium can grow without antibiotics.

Restriction Digestion for to making a low copy plasmid without insert

Name	Sample	10xBuffer	BSA	Enzyme 1		Enzyme 2		MilliQ	Total	Incubation
pSB4K5	2.0 µL	5.0	0.5	XbaI	0.2	SpeI	0.2	42.1	50.0	At 37℃ for 2h

• The concentration of the sample is 56ng/µL.

Ligation and Phosphorylation

After PCR purification, the sample was ligated.

Name	Sample	Ligation High	Total
pSB4K5	1.0 µL	1.0	2.0

Transformation

Name	Sample	Competent Cell	Total	Plate	Incubation	Result
pSB4K5 without insert	2.0 µL	50.0	52.0	LB (Kan+)	09/01 - 09/02

Overnight culture

The ML and the MS were grown at 37℃ for a night (16 hours).

Overnight culture for the makeup test of 08/11, 08/12

Name	Medium	IPTG
ML	Supplemented M9 (Kan+)	0.00 mM
ML		1.00
MS		0.00
MS		1.00

These samples were grown at 37℃ for 16 hours.

Thursday, September 2 By: Tomonori

Culture

OD600 of the overnight culture was measured.

sample name	OD600
ML	1.580
MS	1.582

The overnight culture of ML was diluted in fresh supplemented M9 medium including 3 different concentration of IPTG and that of MS was diluted without IPTG. OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.

ML 0mM	ML 0.01mM	ML 0.1mM	MS1	MS2	MS3
4h	0.119	0.120	0.124	0.130	0.125	0.111
8h	0.974	1.018	0.958	0.848	0.816	0.792
12h	1.127	1.180	1.242	1.262	1.264	1.189
16h	1.027	1.090	1.137	1.172	1.180	1.159
20h	1.103	1.110	1.151	1.233	1.169	1.225

• Measurement at 12h, 16h and 20h was conducted the day after.

We found that the number of the cell had been in steady state at 12h and after.

Culture for the makeup test of 8/11, 8/12

The overnight culture was diluted to 10-2 in the fresh culture and grown for 3hours. OD600 of the culture was measured and samples for measurement of GFP fluorescence were collected.

sample name	OD600
ML(IPTG 1mM)	0.326
ML(IPTG 0mM)	0.329
MS(IPTG 1mM)	0.328
MS(IPTG 0mM)	0.314

Transformation of pSB4K5 without insertion

Colonies were seen on the plate.

Friday, 3 September By:Tomonori, Tasuku

Measure OD600 of the culture of MS and collect samples.

Measure the fluorescence of GFP of the ML and the MS

	ML IPTG 0mM	ML IPTG 0.01mM	ML IPTG 0.1mM	MS1	MS2	MS3
4h	155	392	1836	6526	6572	8274
8h	1046	1667	92602	145341	149358	119017
12h	918	2733	119459	180575	137615	152878
16h	883	2543	80473	138505	159007	235987
20h	868	3229	157578	400829	152590	157942

• Back corrected

We calculate the promoter activity of the lactose promoter

	0mM	0.01mM	0.1mM
4h	0.022	0.055	0.251
8h	0.006	0.010	0.575
12h	0.006	0.018	0.758
16h	0.006	0.015	0.465
20h	0.004	0.015	0.703

• the autofluorescence of E.coli KRX is not corrected.

We could not find the relationship between promoter activity and incubation time.

This graph indicates that the higher IPTG concentration is, the higher the activity of the lactose promoter is.

Sunday, 5 September By Kazuya

Colony PCR of pSB4K5 without insertion

Monday, 6 September By Kazuya

Electrophoresis of pSB4K5 without insertion

Marker	1	2	3	4	5	6	7	8	9	10	11	12	Marker
lambda	pSB4K5 without insertion												100bp

The pSB4K5 must be about 2300bp. The result of the electrophoresis indicates that the transformation was failed.

Overnight culture for measurement the promoter

The ML and the MS were grown at 37℃ for a night (16 hours).

Tuesday, 7 September By: Kazuya, Tomo, Wataru, Fumitaka

Digest pSB4K5 by XbaI, SpeI to make low copy plasmid without insert again

Sample	10xBuffer	BSA	Enzyme1	Enzyme2	MilliQ	Total	Incubation
pSB4K5 2µL	5µL	0.5µL	XbaI 0.2µL	SpeI 0.2µL	42.1µL	50µL	At 37℃2h

• the concentration of the sample, pSB4K5, is 56ng/µL

Ligation and pospholylation

After PCR purification, the sample was ligated.

Sample	Ligation High	Total
pSB4K5 1µL	1µL	2µL

Measure OD600 of the overnight culture

We found that the overnight culture had not grown. We think this is because we added too much antibiotics to the culture.

Wednesday, 8 September By: Tasuku, Wataru

Transformation of pSB4K5 without insert

Sample	Conc(/µL)	Sample Volume(µL)	Competent Cell(µL)	Total	Plate	Incubation
pSB4K5 without insert	-	2	50	52	LB Kan	9/8‾9/9

Overnight culture for measurement the promoter

The ML was grown at 37℃ for a night (18hours).

Thursday, 9 September By:Tasuku

Measure OD600 of the overnight culture

We measure the OD600 of overnight culture. The OD600 is 0.376.

Culture for measurement of Promoter activity

The overnight culture was diluted in fresh supplemented M9 medium including 5 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.

ML	0mM	0.01mM	0.03mM	0.1mM	1mM
4h	0.373	0.325	0.326	0.359	0.341
8h	1.328	1.355	1.481	1.405	1.457

We stopped incubation at 8h because strange floating matters were seen in the culture.

Overnight culture for measurement the promoter

The ML was grown at 37℃ for a night (16hours).

Transformation of pSB4K5 without insert

Colonies were seen on the plate.

Friday, 10 September By: Tasuku

The overnight culture was diluted in fresh supplemented M9 medium including 5 different IPTG concentration and OD600 of each medium was measured every 4 hours. The samples to measure fluorescence of GFP were also collected.

ML	0mM	0.01mM	0.03mM	0.1mM	1mM
4h	0.103	0.109	0.117	0.11	0.055
8h	0.196	0.256	0.285	0.377	0.174

We stopped incubation at 8h because the cell growth rate was too bad.

Sunday, 12 September By: Wataru

Overnight culture of MB

MB (Measurement ? Back), that is, E.coli KRX transformed with pSB4K5 without insert was grown in the supplemented M9 medium for a night (16h).

Monday, 13 September By: Takuya Okada, Ken

Culture for measurement of the promoter activity

OD600 of MB was measured.

4h	8h	12h	16h	20h
0.148	1.738	1.952	1.8	2.178

• Measurements at 12h, 16h and 20h were conducted the day after.

Tuesday, 14 September By: Kazuya, Tomonori

MeasureOD600 of MB culture and collect samples

Measure the fluorescence of the samples

	9/9					9/10					MB
	0mM	0.01mM	0.03mM	0.1mM	1mM	0mM	0.01mM	0.03mM	0.1mM	1mM
4h	2487	3322	4728	23302	69101	225	264	389	3391	5500	77
8h	4472	11295	52260	147947	241965	1761	2139	43379	138454	187563	635
12h											459
16h											382
20h											751

• Back corrected

We measure GFP fluorescence of the samples, but we did not these data because GFP fluorescence of ML samples might have grown abnormally.

Transformation of pSB4K5 without insert　to E.coli BL21 for measurement of lactose degradation

Sample Conc(/µL)	Sample Volume(µL)	Competent Cell(µL)	Total	Plate	Incubation
pSB4K5 without insert	-	2	50	52	LB Kan	9/14‾9/15

Overnight culture of ML for measurement promoter activity

ML in three test tubes was grown for 13hours.

Wednesday, 15 September By: Tomonori

Make the standard curve for measurement lactose concentration

We also measure the concentration of lactose in the supplemented M9 medium and found that it contains approximately 0.01mM lactose.

Thursday, 16 September By: Tomonori

Overnight culture for measurement of RPU of the lactose promoter

We changed the way of measurement of promoter activity. (to know more detail, see protocol.) Three falcon tubes of ML, three falcon tubes of MS and three falcon tubes of MB were grown with 1mM IPTG at 37℃ for 12hours.

Transformation of pSB4K5 without insert to E.coli BL21 for measurement of lactose degradation

Any colony was not seen on the plate.

Friday, 17 September By: Tomonori, Hitoshi, Takuya Okada

Measure OD600 of overnight culture

sample name	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	1.900	1.745	1.937	1.905	1.938	1.488	3.276	3.300	3.202

Culture for measurement of RPU of the lactose promoter

Overnight culture was diluted in the fresh medium including 1mM IPTG and OD600 was measured at 1h, 2h, 3h, 3.5h, 4h.

OD600	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
1h	0.017	0.103	0.118	0.057	0.073	0.044	-0.028	0.101	0.098
2h	0.270	0.295	0.245	0.167	0.241	0.149	0.208	0.179	0.294
3h	0.33	0.33	0.35	0.36	0.42	0.31	0.66	0.70	0.66
3.5h	0.47	0.37	0.46	0.49	0.55	0.40	0.98	0.98	0.96
4h	0.675	0.595	0.665	0.684	0.793	0.607	1.358	1.362	1.360

This graph indicates that all samples were in the exponential growth from 2h to 4h. The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.

GFP	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
3h	58603	49991	50845	53416	55463	40321	204	167	152
3.5h	89428	76573	85433	67457	83329	59463	218	271	246

Back corrected We calculate the RPU of the lactose promoter with 1mM IPTG is 1.65.

	ML-A	ML-B	ML-C	average	stdev	CV
RPU	1.60	1.58	1.77	1.65	0.106	0.0642

Overnight culture of the ML was also diluted in the fresh medium including 0mM IPTG, 0.01mM IPTG, 0.03mM IPTG, 0.1mM IPTG and 1mM IPTG and the samples for measurement of GFP fluorescence were collected at 3h and 3.5h.

IPTG 0mM	ML-A	ML-B	ML-C
3h	1906	2138	2190
3.5h	1781	1141	980
IPTG 0.01mM	ML-A	ML-B	ML-C
3h	5037	2588	2189
3.5h	1191	1700	1511
IPTG 0.03mM	ML-A	ML-B	ML-C
3h	2525	2410	3065
3.5h	1982	3330	3723
IPTG 0.1mM	ML-A	ML-B	ML-C
3h	14191	9154	9856
3.5h	6897	5427	7084

• Back corrected

We did not calculate RPU from these data because GFP fluorescence at 3.5h was lower than that at 3h in most of the samples, and we couldn't calculate GFP synthesis rate. We think this is because GFP synthesized in overnight culture with IPTG 1mM had stayed more at 3h than 3.5h.

Make a new plate of the MB

Monday, 20 September By: Tomonori

Make the supplemented M9 medium without casamino acids

We think that lactose in the supplemented M9 medium come from casamino acids, so we decide to measure RPU of the lactose promoter without casamino acids because lactose in medium induces the lactose promoter.

Overnight culture for measurement of RPU of the lactose promoter with IPTG 0.1mM

Three falcon tubes of the ML, three falcon tubes of the MS and three falcon tubes of the MB were grown in the supplemented M9 medium without casamino acids including 0.1mM IPTG at 37℃ for 23 hours.

Tuesday, 21 September By: Hitoshi

Measure OD600 of overnight culture

sample name	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	2.140	2.080	2.103	1.975	1.964	1.993	0.041	0.062	0.025

We found that MB had not grown.

Culture for measurement of RPU of the lactose promoter

ML and MS were cultured and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h in the medium without casamino acids. The samples were collected at 3h and 3,5h.

OD600 IPTG 0.1mM</br>	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
2h	-0.056	-0.002	0.004	0.027	-0.001	0.024
2.5h	-0.023	0.000	0.014	-0.050	0.003	0.0025
3h	-0.050	0.003	0.0025	0.0036	0.009	0.040
3.5h	-0.036	0.002	0.041	0.061	0.061	0.039
4h	-0.030	0.045	0.04	0.0175	0.074	0.104

We found that ML and MS had grown hardly.

Overnight culture for measurement of RPU of the lactose promoter with IPTG 0mM

Three falcon tubes of ML, three falcon tubes of MS and three falcon tubes of MB were grown in the supplemented M9 medium without casamino acids including 0mM IPTG and 0.01mM ITPG at 37℃ for 23 hours.

Wednesday, 22 September By: Hitoshi, Takuya Okada

Measure OD600 of overnight culture

IPTG 0mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	2.009	1.948	1.965	2.168	1.951	1.719	0.042	0.052	0.032
IPTG 0.01mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	2.091	1.414	2.082	1.460	1.959	2.099	0.035	0.050	0.042

We found that MB had not grown.

Culture for measurement of RPU of the lactose promoter

ML and MS were cultured and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h in the medium without casamino acids. The samples were collected at 3h and 3,5h.

OD600 IPTG 0mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
2h	0.099	0.102	0.119	0.080	0.082	0.068
2.5h	0.115	0.141	0.136	0.092	0.068	0.110
3h	0.176	0.173	0.193	0.130	0.093	0.124
3.5h	0.236	0.237	0.248	0.201	0.129	0.169
4h	0.320	0.249	0.256	0.243	0.156	0.232
OD600 IPTG 0.01mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
2h	0.135	0.070	0.116	0.060	0.075	0.092
2.5h	0.155	0.091	0.142	0.071	0.103	0.127
3h	0.167	0.092	0.196	0.080	0.148	0.168
3.5h	0.227	0.180	0.331	0.151	0.230	0.271

We found that cell growth rate was low without casamino acids and that OD600 of the culture at 3h was low, therefore we decide to measure OD600 at 4h, 4.5h, 5h, 5.5h and 6h, and to collect samples at 5h and 5.5h in later experiment without casamino acids.

Overnight culture for the measurement of lactose promoter

We cultivated ML and MS in the supplemented M9 medium without casamino acids including 0.03mM IPTG and 0.1mM IPTG.

Thursday, 23 September By: Hitoshi, Kazuya

Measure OD600 of the overnight culture

0.03mM	ML	MS
OD600	1.417	1.483
0.1mM	ML	MS
OD600	2.126	2.131

Culture for measurement of RPU of the lactose promoter

ML and MS were cultured and OD600 was measured at 4h, 4.5h, 5h, 5.5h and 6h in the medium without casamino acids. The samples were collected at 5h and 5.5h.

0.03mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
4h	0.266	0.256	0.273	0.208	0.188	0.267
4.5h	0.345	0.316	0.360	0.280	0.232	0.213
5h	0.506	0.456	0.531	0.337	0.309	0.270
5.5h	0.580	0.569	0.623	0.380	0.330	0.334
6h	0.764	0.699	0.775	0.415	0.396	0.395

0.1mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
4h	0.237	0.259	0.26	0.279	0.282	0.286
4.5h	0.325	0.339	0.348	0.429	0.423	0.419
5h	0.442	0.455	0.485	0.563	0.541	0.572
5.5h	0.602	0.599	0.615	0.749	0.675	0.682
6h	0.719	0.678	0.741	0.987	0.815	0.900

Overnight culture for the measurement of lactose promoter

We cultivated ML and MS in the supplemented M9 medium without casamino acids including 1mM IPTG.

Friday, 24 September By: Tomonori, Hitoshi, Tasuku, Takuya Okada, Ken, Kazuya

Measure OD600 of the overnight culture

1mM	ML	MS
OD600	1.187	1.427

Culture for measurement of RPU of the lactose promoter

ML and MS were cultured and OD600 was measured at 4h, 4.5h, 5h, 5.5h and 6h in the medium without casamino acids. The samples were collected at 5h and 5.5h.

1mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
4h	0.127	0.11	0.107	0.168	0.169	0.208
4.5h	0.161	0.153	0.153	0.255	0.254	0.275
5h	0.220	0.206	0.209	0.366	0.353	0.383
5.5h	0.349	0.324	0.293	0.479	0.483	0.501
6h	0.439	0.431	0.384	0.628	0.651	0.645

Measure the fluorescence of GFP of the ML and the MS

0mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
3h	292	263	88	6086	3007	6443
3.5h	131	93	-45	1700	1676	1652
0.01mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
3h	124	42	378	3425	6640	5990
3.5h	330	-41	632	5830	10515	13601
0.03mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
5h	48848	23900	51150	38837	13416	33454
5.5h	54980	54980	54980	35758	34595	34443
0.1mM 20 September</br>	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
3h	31	3327	2219	3331	2298	3766
3.5h	61	5341	3489	4840	3955	4512
0.1mM 23 September</br>	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C
5h	72902	68382	78054	56365	42563	57917
5.5h	98103	84737	81525	89239	91295	68302

Back corrected We can't use the data of IPTG 0mM to calculate RPU because GFP fluorescence at 3.5h is lower than that at 3h. Also we can't use the data of ML-B 0.01mM IPTG and the data of MS-A of 0.03mM. Although GFP fluorescence of 0.1mM (20 September) at 3.5h is higher than that at 3h, OD600 is so low. Accordingly, We can't use the data of 0.1mM (20 September).

Overnight culture for the measurement of lactose promoter

Three tubes of the ML, three tubes of the MS and three tubes of the MB were grown with casamino acids including 0.1mM ITPG and 0.01mM IPTG for 16hours.

Saturday, 25 September By: Hitoshi, Tomonori

Measure OD600 of the overnight culture

0.1mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	1.961	1.823	2.078	2.241	2.383	1.857	2.674	2.674	2.632
0.01mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	1.754	1.868	1.702	1.885	2.009	1.940	2.693	2.631	2.622

Culture for measurement of RPU of the lactose promoter

Overnight culture was diluted in the fresh medium and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h. The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.

0.1mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
2h	0.186	0.184	0.217	0.191	0.208	0.204	0.347	0.298	0.311
2.5h	0.289	0.302	0.334	0.289	0.313	0.315	0.624	0.542	0.567
3h	0.408	0.44	0.441	0.432	0.426	0.406	0.947	0.843	0.858
3.5h	0.519	0.584	0.602	0.558	0.6	0.539	1.33	1.21	1.25
4h	0.784	0.805	0.893	0.777	0.848	0.743	1.655	1.561	1.582

Overnight culture for the measurement of lactose promoter

Three tubes of the ML, three tubes of the MS and three tubes of the MB were grown with casamino acids including 0mM ITPG.

Sunday, 26 September By: Tomonori

Measure OD600 of the overnight culture

0mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	1.646	1.576	1.643	1.702	1.832	1.826	2.272	2.090	1.985

Culture for measurement of RPU of the lactose promoter

Overnight culture was diluted in the fresh medium and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h. The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.

0mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
2h	0.289	0.195	0.247	0.213	0.237	0.243	0.289	0.265	0.229
2.5h	0.365	0.317	0.307	0.278	0.316	0.316	0.387	0.358	0.428
3h	0.385	0.295	0.31	0.344	0.349	0.332	0.651	0.578	0.642
3.5h	0.456	0.519	0.493	0.453	0.464	0.49	0.859	0.885	0.883
4h	0.65	0.659	0.645	0.567	0.565	0.642	1.215	1.189	1.199

Overnight culture for the measurement of lactose promoter

Three tubes of the ML, three tubes of the MS and three tubes of the MB were grown with casamino acids including 0.03mM ITPG.

Monday, 27 September By: Tomonori, Tasuku, Ken

Measure OD600 of the overnight culture

0.03mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	1.646	1.576	1.643	1.702	1.832	1.826	2.272	2.090	1.985

Culture for measurement of RPU of the lactose promoter

Overnight culture was diluted in the fresh medium and OD600 was measured at 2h, 2.5h, 3h, 3.5h and 4h. The samples for measurement of GFP fluorescence were collected at 3h and 3.5h.

0.03mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
2h	0.214	0.239	0.218	0.238	0.232	0.217	0.376	0.383	0.315
2.5h	0.343	0.307	0.329	0.318	0.258	0.316	0.583	0.541	0.554
3h	0.43	0.373	0.389	0.39	0.358	0.406	0.773	0.803	0.746
3.5h	0.509	0.446	0.538	0.46	0.488	0.548	1.148	1.114	1.054
4h	0.637	0.635	0.709	0.626	0.616	0.74	1.524	1.479	1.385

Measure the GFP fluorescence

0mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
3h	589	508	417	43120	40563	50343	-368	230	255
3.5h	704	938	868	58066	66566	65910	346	765	508
0.01mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
3h	1187	1316	1329	59806	69376	62702	360	396	439
3.5h	1883	1707	1507	96634	93095	88262	583	559	588
0.03mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
3h	2341	884	2575	34995	33056	14529	273	171	268
3.5h	3219	3124	1243	18855	20536	14040	416	230	209
0.1mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
3h	17202	20387	25090	61144	13667	54117	313	401	388
3.5h	22040	25372	32237	87691	89368	76786	613	338	476

Back corrected We did not use the data in grey cells of the above table.

RPU	ML-A	ML-B	ML-C	average	stdev	CV
0mM	-0.00062	0.0122	0.0147	0.0134	0.00821	0.611
0.01mM	0.0191	0.00963	0.00244	0.0144	0.00838	0.583
0.1mM	0.205	0.191	0.269	0.221	0.0415	0.188

We did not use the data in grey cells of the above table. CV of RPU of 0mM IPTG and that of 0.01mM IPTG is high. In addition, the supplemented M9 medium used in the experiment includes 0.01mM lactose. The RPU of the lactose promoter induced by IPTG only must be lower than the data resulted from this experiment.

Sunday, 3 October By: Ken

Overnight culture for measurement of lactose promoter

ML, MS and MB is cultivated in 5mL the supplemented M) medium with casamino acids and 0.5mM IPTG at 37℃ for a night.

Culture for measurement of lactose promoter

The overnight culture was diluted in the fresh medium and OD60 was measured at 3h and 3.5h. The samples for measurement GFP fluorescence are also collected at 3h and 3.5h.

OD600	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
3h	0.765	0.819	0.713	0.694	0.877	0.776	0.672	1.25	1.262
3.5h	1.113	1.42	1.07	1.316	1.531	1.326	1.699	1.986	1.963

GFP fluorescence	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
3h	38234	42544	29945	83245	83303	84562	458	382	523
3.5h	56290	71568	56553	160709	172546	156292	570	687	688

RPU	ML-A	ML-B	ML-C	average	stdev	CV
0.5mM	0.26287742	0.353636125	0.408065404	0.341526316	0.07334762	0.214764181

Thursday, 14 October By: Ken

Overnight culture for measurement of lactose promoter

We cultivate ML, MS and MB in 5mL the supplemented M9 medium with casamino acids and 0.5mM IPTG at 30℃ for a night.

Fryday, 15 October By: Ken, Tomonori

Measure OD600 of the overnight culture

0.5mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	2.268	2.2	2.169	1.236	2.964	1.793	2.037	1.701	2.194

Culture for measurement of lactose promoter

Overnight culture was diluted in the fresh medium and OD600 was measured at 3h and 3.5h. However, the samples for measurement GFP fluorescence are collected at 3h and 3.5h, we could not collect enough amount of bacterium because OD600 of the culture was low. We did not measure GFP fluorescence.

OD600	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	0.141	0.145	0.124	0.158	0.15	0.163	0.162	0.178	0.141
3.5h	0.178	0.22	0.209	0.198	0.164	0.211	0.199	0.234	0.178

Overnight culture for measurement of lactose promoter

We cultivate ML, MS and MB in 5mL the supplemented M9 medium without casamino acids and 0.5mM IPTG at 37℃ for a night. Measure the lactose concentration in the supplemented M9 medium again

samples	0mM lactose	0.004mM lactose	0.008mM lactose	0.012mM lactose	0.016mM lacotse	0.02mM lactose	M9+	M9+	M9+	M9-	M9-	M9-
fluorescence	57903	30132	49578	42397	50601	38140	51195	49293	77840	59711	62300	66276

• The sample M9+ is the supplemented M9 medium with casamino acids and the sample M9- is the supplemented M9 medium without casamino acids.

The fluorescence of all samples is too low compared to the result of the measurement at first time (15 September). We failed to measure the lactose concentration in the supplemented M9 medium.

Saturday, 16 October By: Tomonori, Ken

Measure the lactose promoter activity with the 0.5mM IPTG

The overnight culture did not grow.

Measure the lactose concentration in the supplemented M9 medium again

samples	0mM lactose	0.004mM lactose	0.008mM lactose	0.012mM lactose	0.016mM lacotse	0.02mM lactose	M9+	M9+	M9+	M9-	M9-	M9-
Fluorescence	51805	49721	53813	57170	82148	108739	74975	75924	63856	64537	102611	89683

The fluorescence of all samples is too low compared to the result of the measurement at first time (15 September) again.

Overnight culture for lactose promoter activity

We cultivated ML, MS and MB in 5mL the supplemented M9 medium with casamino acids and 0.5mM IPTG at 37℃ for a night. We also cultivated ML, MS and MB in 5mL the supplemented M9 medium without casamino acids and 0.5mM IPTG at 37℃ for a night.

Sunday, 17 October By: Tomonori, Ken

Culture for the measurement of lactose promoter acitivty

The overnight culture not containing casamino acids did not grow. Only the overnight culture containing casamino acids is diluted in the fresh medium and grown. At 1h, 1.5h, 2h, 2.5h, 3h after dilution, OD600 of the culture was measured. The culture was also collected at 3h and 3,5h after dilution to measure GFP fluorescene.

OD600 MB_C	1h	1.5h	2h	2.5h	3h
	0.200	0.308	0.455	0.588	0.765

OD600	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
1.5h	0.219	0.239	0.207	0.221	0.230	0.218	0.203	0.205	0.215
2h	0.358	0.330	0.375	0.370	0.372	0.357	0.363	0.367	0.378

GFP fluorescene	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
1.5h	8379	6161	4563	4806	4291	6448	46	-15	-65	2h	11718	18965	20988	7415	6519	7073	-40	28	49

sample	MLA	MLB	MLC
RPU	1.426109	5.545562	6.954958

We failed to the measurement because RPU of MLB and MLC are so high. The overnight culture containing casamino acids is diluted in the fresh medium and grown again. At 2h and 2.5h after dilution, OD600 of the culture was measured and the samples for GFP fluorescence were collected.

OD600	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
2h	0.498	0.476	0.457	0.448	0.485	0.6	0.345	0.351	0.371
2.5h	0.723	0.64	0.645	0.709	0.688	0.554	0.639	0.656	0.658

GFP fluorescence	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
2h	8208	4927	17585	9711	4847	8952	-2	-46	-16
2.5h	15567	7190	6937	7762	7169	6212	-14	249	5

GFP fluorescence of some samples did not increase, and we noticed that pellet of E.coli did not resolve enough. We found that cell lysis is not required to measure the GFP fluorescence quantitatively, We decided not to make cell pellet resolved in measurement of GFP fluorescence.

Overnight culture for lactose promoter activity

We cultivated ML, MS and MB in 5mL the supplemented M9 medium with casamino acids and 0.8mM IPTG at 37℃ for a night.

Monday, 18 Ocotober By: Ken

Measure OD600 of the overnight culture

0.8mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	1.646	1.576	1.643	1.702	1.832	1.826	2.272	2.090	1.985

Culture for measurement of lactose promoter

The overnight culture was diluted in fresh medium and OD600 was measured after 3h and 3.5h. The samples for measurement of GFP fluorescence were also collecte at 3h and 3.5h after the dilution.

OD600	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	0.541	0.59	0.562	0.616	0.596	0.562	0.486	0.498	0.46
3.5h	0.803	0.82	0.817	0.921	0.872	0.84	0.806	0.811	0.796

GFP fluorescence	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	133519	133447	117828	115401	125899	112488	859	842	773
3.5h	178972	208432	157171	160958	151790	145594	1813	1563	1595

RPU	ML-A	ML-B	ML-C	average	stdev	CV
0.8mM	1.44	2.28	1.21	1.33	0.161	0.122

CV is low, so the measurement was success. Overnight culture for lactose promoter activity We cultivated ML, MS and MB in 5mL the supplemented M9 medium with casamino acids and 2mM IPTG at 37℃ for a night.

Tuesday, 19 October By: Ken

Measure OD600 of the overnight culture

2mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	3.067	3.067	3.067	2.936	2.936	2.936	2.322	2.322	2.322

Culture for measurement of lactose promoter

The overnight culture was diluted in fresh medium and OD600 was measured after 3h and 3.5h. The samples for measurement of GFP fluorescence were also collecte at 3h and 3.5h after the dilution.

OD600	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	0.42	0.437	0.417	0.475	0.438	0.484	0.438	0.408	0.419
3.5h	0.574	0.628	0.593	0.652	0.644	0.707	0.682	0.631	0.653

GFP fluorescence	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	88857	82223	86475	86545	76677	85028	758	661	695
3.5h	124316	128976	120154	108873	107828	113082	1103	981	1061

RPU	ML-A	ML-B	ML-C	average	stdev	CV
2mM	1.4899	1.834	1.3908	1.5716	0.2326	0.148

Wednesday, 20 October By: Ken

Overnight culture for lactose promoter activity

We cultivated ML, MS and MB in 5mL the supplemented M9 medium with casamino acids and 0.3mM IPTG at 37℃ for a night. We also cultivated ML, MS and MB in 5mL the supplemented M9 medium with casamino acids and 0.03mM IPTG at 37℃ for a night.

Thursday, 21 October By: Ken

Measure OD600 of the overnight culture

0.3mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	3.099	3.099	3.099	3.14	3.14	3.14	3.181	3.181	3.181

0.03mM	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	3.192	3.192	3.192	3.18	3.18	3.18	3.247	3.247	3.247

Culture for the measurement of the lactose promoter activity

The overnight culture was diluted in fresh medium and OD600 was measured after 3h and 4h. The samples for measurement of GFP fluorescence were also collecte at 3h and 4h after the dilution.

OD600 IPTG 0.3mM	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	0.5	0.492	0.502	0.543	0.499	0.522	0.634	0.615	0.63
4h	0.83	0.85	0.878	0.912	0.855	0.893	1.327	1.277	1.318

GFP fluorescence IPTG 0.3mM	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	105341	94859	101571	106905	94079	110943	1115	1039	1007
4h	153111	143951	152096	170446	149843	153449	2013	1851	1883

RPU	ML-A	ML-B	ML-C	average	stdev	CV
0.3mM	0.9378	0.9545	0.9551	0.9491	0.0098	0.0103

OD600 IPTG 0.03mM	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	0.505	0.499	0.517	0.519	0.501	0.528	0.623	0.651	0.631
4h	0.903	0.861	0.909	0.889	0.881	0.909	1.276	1.352	1.319

GFP fluorescence IPTG 0.03mM	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	33137	28000	34213	101336	91771	88778	1057	1173	999
4h	47691	40370	49962	190889	167666	163445	1899	2081	1976

RPU	ML-A	ML-B	ML-C	average	stdev	CV
0.03mM	0.17661	0.15511	0.18935	0.17369	0.01731	0.09966

Sunday, 24 October By: Ken

Overnight culture for lactose promoter activity

We cultivated ML, MS and MB in 5mL the supplemented M9 medium with casamino acids and 0.5mM IPTG at 37℃ for a night.

Monday, 25 October By: Ken

Measure OD600 of the overnight culture

	ML-A	ML-B	ML-C	MS-A	MS-B	MS-C	MB-A	MB-B	MB-C
OD600	3.153	3.153	3.153	2.905	2.905	2.905	3.18	3.18	3.18

Culture for the measurement of the lactose promoter activity

The overnight culture was diluted in fresh medium and OD600 was measured after 3h and 4h. The samples for measurement of GFP fluorescence were also collecte at 3h and 4h after the dilution.

OD600 IPTG 0.3mM	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	0.549	0.546	0.542	0.51	0.519	0.512	0.847	0.813	X
3.5h	0.723	0.726	0.718	0.678	0.668	0.667	1.226	1.191	1.195

GFP fluorescence IPTG 0.3mM	MLA	MLB	MLC	MSA	MSB	MSC	MBA	MBB	MBC
3h	33137	28000	34213	101336	91771	88778	1057	1173	999
3.5h	47691	40370	49962	190889	167666	163445	1899	2081	1976

RPU	ML-A	ML-B	ML-C	average	stdev	CV
0.03mM	0.165	0.1393	0.1806	0.1616	0.0209	0.1291

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