Team:Kyoto/Notebook2

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Contents

Notebook2

Measurement of R0011

Wednesday, August 11 By: Wataru

Overnight culture
NameStrainMediumInducer(IPTG)Incubation
ML-KRXLB (Kan+), 3mL0mMAt 37℃ 20:20- (Overnight)
ML+KRX1mM
MS-KRX0mM
MS+KRX1mM
KRXKRXSOC, 3mL0mM

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.

NameOptical Density (600nm)Fluorescence (485/535nm)Calculation 1Calculation 2Calculation 3Promoter Activity
ML-0.531488146978.85 × 1036.40 × 1038.65 × 10-2
ML+0.54691549913651.67 × 1051.65 × 1052.04
MS-0.64549469492857.64 × 1047.40 × 1041 (0mM IPTG)
MS+0.62352017518338.32 × 1048.08 × 1041 (1mM IPTG)
KRX1.413364434602.449 × 103--
Cell Lysis Reagent-184----
  • Calculation 1: Fluorescence - FluorescenceCell Lysis Reagent
  • Calculation 2: Calculation 1 / Optical Density (600nm)
  • Calculation 3: Calculation 2 - Calculation 2KRX
  • Promoter Activity: Calculation 3ML- / Calculation 3MS-, Calculation 3ML+ / Calculation 3MS+


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 BBa_K358000 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.

0.00mM0.010.030.050.070.101.00
0h-0.0030.0140.000-0.008---
4h0.0010.1000.0330.062---
6h----0.1180.1870.178
8h0.2270.4020.3630.3460.3600.4790.517
161.1171.1421.1311.2371.2111.2021.156
20h1.1071.1181.0841.1781.1771.1781.131
  • 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.
KyotoGrp100825-1.png

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 BBa_I20260 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.

Time0.00mM0.010.030.050.070.101.00
0h-0.016-0.0150.000-0.016-0.0180.001-0.007
40.1080.1120.1160.0940.1270.1160.160
80.6260.5850.5910.5220.5160.5620.553
161.3511.4251.4421.4831.3461.4201.497
201.3631.4051.4031.4421.3831.3611.434
24-1.297----1.352
  • Measurement at 16h, 20h, and 24h was conducted the day after.
KyotoGrp100826-1.png
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.

NameOD600
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.

NameOD600
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
Time0.00mM0.010.030.050.070.101.00
0h51329576268405
4126161213046431726061367712120
836181742926703265566428507876168736
165573727447593526613128443091
201229299015123189357979192437
  • Back corrected.
MS
Time0.00mM0.010.030.050.070.101.00
0h3-13-11165632-14
41763877901341380561963280908800
866508851002363198345912651517732384395
1665047556615976504228424010662
2078351163890701513515383868118276
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.

Time0.00mM0.010.030.050.070.101.00
0h-------
40.0000000.8800410.7982670.8124540.1428671.0487181.237998
81.5002211.2704050.4618111.1852230.4854630.5211310.871168
161.0363601.2012410.9851630.5525761.6117480.7924060.375426
200.1931350.3228700.2157610.2579240.2733801.0539340.169068
  • The autofluorescence of E.coli KRX is not corrected.
KyotoGrp100831-1.png
KyotoGrp100831-2.png

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.

NameOD600
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
NameSample10xBufferBSAEnzyme 1Enzyme 2MilliQTotalIncubation
pSB4K52.0 µL5.00.5XbaI0.2SpeI0.242.150.0At 37℃ for 2h
  • The concentration of the sample is 56ng/µL.
Ligation and Phosphorylation

After PCR purification, the sample was ligated.

NameSampleLigation HighTotal
pSB4K51.0 µL1.02.0
Transformation
NameSampleCompetent CellTotalPlateIncubationResult
pSB4K5 without insert2.0 µL50.052.0LB (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
NameMediumIPTG
MLSupplemented M9 (Kan+)0.00 mM
ML1.00
MS0.00
MS1.00

These samples were grown at 37℃ for 16 hours.


Thursday, September 2 By: Tomonori

Culture

OD600 of the overnight culture was measured.

sample nameOD600
ML1.580
MS1.582

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 0mMML 0.01mMML 0.1mMMS1MS2MS3
4h0.1190.1200.1240.1300.1250.111
8h0.9741.0180.9580.8480.8160.792
12h1.1271.1801.2421.2621.2641.189
16h1.0271.0901.1371.1721.1801.159
20h1.1031.1101.1511.2331.1691.225
  • Measurement at 12h, 16h and 20h was conducted the day after.
KyotoGrp100902-1.png

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 nameOD600
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 0mMML IPTG 0.01mMML IPTG 0.1mMMS1MS2MS3
4h1553921836652665728274
8h1046166792602145341149358119017
12h9182733119459180575137615152878
16h883254380473138505159007235987
20h8683229157578400829152590157942
  • Back corrected

We calculate the promoter activity of the lactose promoter

0mM0.01mM0.1mM
4h0.0220.0550.251
8h0.0060.0100.575
12h0.0060.0180.758
16h0.0060.0150.465
20h0.0040.0150.703
  • the autofluorescence of E.coli KRX is not corrected.
KyotoGrp100903-1.png

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

KyotoGrp100903-2.png

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
Marker123456789101112Marker
lambdapSB4K5 without insertion100bp

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

KyotoExp100906-1.png
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
Sample10xBufferBSAEnzyme1Enzyme2MilliQTotalIncubation
pSB4K5 2µL5µL0.5µLXbaI 0.2µLSpeI 0.2µL42.1µL50µLAt 37℃2h
  • the concentration of the sample, pSB4K5, is 56ng/µL
Ligation and pospholylation

After PCR purification, the sample was ligated.

SampleLigation HighTotal
pSB4K5 1µL1µL2µL
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
SampleConc(/µL)Sample Volume(µL)Competent Cell(µL)TotalPlateIncubation
pSB4K5 without insert-25052LB Kan9/8‾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.

ML0mM0.01mM0.03mM0.1mM1mM
4h0.3730.3250.3260.3590.341
8h1.3281.3551.4811.4051.457

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.

ML0mM0.01mM0.03mM0.1mM1mM
4h0.1030.1090.1170.110.055
8h0.1960.2560.2850.3770.174

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.

4h8h12h16h20h
0.1481.7381.9521.82.178
  • 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
9/99/10MB
0mM0.01mM0.03mM0.1mM1mM0mM0.01mM0.03mM0.1mM1mM
4h24873322472823302691012252643893391550077
8h447211295522601479472419651761213943379138454187563635
12h459
16h382
20h751
  • 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)TotalPlateIncubation
pSB4K5 without insert-25052LB Kan9/14‾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
KyotoGrp100915-1.png

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 nameML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6001.9001.7451.9371.9051.9381.4883.2763.3003.202
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.

OD600ML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
1h0.0170.1030.1180.0570.0730.044-0.0280.1010.098
2h0.2700.2950.2450.1670.2410.1490.2080.1790.294
3h0.330.330.350.360.420.310.660.700.66
3.5h0.470.370.460.490.550.400.980.980.96
4h0.6750.5950.6650.6840.7930.6071.3581.3621.360
KyotoGrp100917-1.png

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.

GFPML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
3h586034999150845534165546340321204167152
3.5h894287657385433674578332959463218271246

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

ML-AML-BML-CaveragestdevCV
RPU1.601.581.771.650.1060.0642

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 0mMML-AML-BML-C
3h190621382190
3.5h17811141980
IPTG 0.01mMML-AML-BML-C
3h503725882189
3.5h119117001511
IPTG 0.03mMML-AML-BML-C
3h252524103065
3.5h198233303723
IPTG 0.1mMML-AML-BML-C
3h1419191549856
3.5h689754277084
  • 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 nameML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6002.1402.0802.1031.9751.9641.9930.0410.0620.025

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-AML-BML-CMS-AMS-BMS-C
2h-0.056-0.0020.0040.027-0.0010.024
2.5h-0.0230.0000.014-0.0500.0030.0025
3h-0.0500.0030.00250.00360.0090.040
3.5h-0.0360.0020.0410.0610.0610.039
4h-0.0300.0450.040.01750.0740.104

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 0mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6002.0091.9481.9652.1681.9511.7190.0420.0520.032
IPTG 0.01mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6002.0911.4142.0821.4601.9592.0990.0350.0500.042

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 0mMML-AML-BML-CMS-AMS-BMS-C
2h0.0990.1020.1190.0800.0820.068
2.5h0.1150.1410.1360.0920.0680.110
3h0.1760.1730.1930.1300.0930.124
3.5h0.2360.2370.2480.2010.1290.169
4h0.3200.2490.2560.2430.1560.232
OD600 IPTG 0.01mMML-AML-BML-CMS-AMS-BMS-C
2h0.1350.0700.1160.0600.0750.092
2.5h0.1550.0910.1420.0710.1030.127
3h0.1670.0920.1960.0800.1480.168
3.5h0.2270.1800.3310.1510.2300.271
KyotoGrp100922-1.png
KyotoGrp100922-2.png

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
0.03mMMLMS
OD6001.4171.483
0.1mMMLMS
OD6002.1262.131
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.

0.03mMML-AML-BML-CMS-AMS-BMS-C
4h0.2660.2560.2730.2080.1880.267
4.5h0.3450.3160.3600.2800.2320.213
5h0.5060.4560.5310.3370.3090.270
5.5h0.5800.5690.6230.3800.3300.334
6h0.7640.6990.7750.4150.3960.395
KyotoGrp100923-1.png
0.1mMML-AML-BML-CMS-AMS-BMS-C
4h0.2370.2590.260.2790.2820.286
4.5h0.3250.3390.3480.4290.4230.419
5h0.4420.4550.4850.5630.5410.572
5.5h0.6020.5990.6150.7490.6750.682
6h0.7190.6780.7410.9870.8150.900
KyotoGrp100923-2.png
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
1mMMLMS
OD6001.1871.427
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.

1mMML-AML-BML-CMS-AMS-BMS-C
4h0.1270.110.1070.1680.1690.208
4.5h0.1610.1530.1530.2550.2540.275
5h0.2200.2060.2090.3660.3530.383
5.5h0.3490.3240.2930.4790.4830.501
6h0.4390.4310.3840.6280.6510.645
KyotoGrp100924-1.png
Measure the fluorescence of GFP of the ML and the MS
0mMML-AML-BML-CMS-AMS-BMS-C
3h29226388608630076443
3.5h13193-45170016761652
0.01mMML-AML-BML-CMS-AMS-BMS-C
3h12442378342566405990
3.5h330-4163258301051513601
0.03mMML-AML-BML-CMS-AMS-BMS-C
5h488482390051150388371341633454
5.5h549805498054980357583459534443
0.1mM
20 September</br>
ML-AML-BML-CMS-AMS-BMS-C
3h3133272219333122983766
3.5h6153413489484039554512
0.1mM
23 September</br>
ML-AML-BML-CMS-AMS-BMS-C
5h729026838278054563654256357917
5.5h981038473781525892399129568302

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
0.1mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6001.9611.8232.0782.2412.3831.8572.6742.6742.632
0.01mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6001.7541.8681.7021.8852.0091.9402.6932.6312.622
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.

0.1mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
2h0.1860.1840.2170.1910.2080.2040.3470.2980.311
2.5h0.2890.3020.3340.2890.3130.3150.6240.5420.567
3h0.4080.440.4410.4320.4260.4060.9470.8430.858
3.5h0.5190.5840.6020.5580.60.5391.331.211.25
4h0.7840.8050.8930.7770.8480.7431.6551.5611.582
KyotoGrp100925-1.png
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
0mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6001.6461.5761.6431.7021.8321.8262.2722.0901.985
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.

0mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
2h0.2890.1950.2470.2130.2370.2430.2890.2650.229
2.5h0.3650.3170.3070.2780.3160.3160.3870.3580.428
3h0.3850.2950.310.3440.3490.3320.6510.5780.642
3.5h0.4560.5190.4930.4530.4640.490.8590.8850.883
4h0.650.6590.6450.5670.5650.6421.2151.1891.199
KyotoGrp100926-1.png
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
0.03mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6001.6461.5761.6431.7021.8321.8262.2722.0901.985
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.

0.03mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
2h0.2140.2390.2180.2380.2320.2170.3760.3830.315
2.5h0.3430.3070.3290.3180.2580.3160.5830.5410.554
3h0.430.3730.3890.390.3580.4060.7730.8030.746
3.5h0.5090.4460.5380.460.4880.5481.1481.1141.054
4h0.6370.6350.7090.6260.6160.741.5241.4791.385
KyotoGrp100927-1.png
Measure the GFP fluorescence
0mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
3h589508417431204056350343-368230255
3.5h704938868580666656665910346765508
0.01mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
3h118713161329598066937662702360396439
3.5h188317071507966349309588262583559588
0.03mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
3h23418842575349953305614529273171268
3.5h321931241243188552053614040416230209
0.1mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
3h172022038725090611441366754117313401388
3.5h220402537232237876918936876786613338476

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

RPUML-AML-BML-CaveragestdevCV
0mM-0.000620.01220.01470.01340.008210.611
0.01mM0.01910.009630.002440.01440.008380.583
0.1mM0.2050.1910.2690.2210.04150.188

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.

OD600ML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
3h 0.7650.8190.7130.6940.8770.7760.6721.251.262
3.5h 1.1131.421.071.3161.5311.3261.6991.9861.963
GFP fluorescenceML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
3h 382344254429945832458330384562458382523
3.5h 562907156856553160709172546156292570687688
RPUML-AML-BML-CaveragestdevCV
0.5mM 0.262877420.3536361250.4080654040.3415263160.073347620.214764181

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
0.5mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6002.2682.22.1691.2362.9641.7932.0371.7012.194
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.

OD600MLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 0.1410.1450.1240.1580.150.1630.1620.1780.141
3.5h 0.1780.220.2090.1980.1640.2110.1990.2340.178
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 lactose0.004mM lactose0.008mM lactose0.012mM lactose0.016mM lacotse0.02mM lactoseM9+M9+M9+M9-M9-M9-
fluorescence 579033013249578423975060138140511954929377840597116230066276
  • 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 lactose0.004mM lactose0.008mM lactose0.012mM lactose0.016mM lacotse0.02mM lactoseM9+M9+M9+M9-M9-M9-
Fluorescence 51805497215381357170821481087397497575924638566453710261189683

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_C1h1.5h2h2.5h3h
0.2000.3080.4550.5880.765
OD600MLAMLBMLCMSAMSBMSCMBAMBBMBC
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 fluoresceneMLAMLBMLCMSAMSBMSCMBAMBBMBC
1.5h 83796161456348064291644846-15-65 2h 117181896520988741565197073-402849
sample MLAMLBMLC
RPU 1.4261095.5455626.954958

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.

OD600MLAMLBMLCMSAMSBMSCMBAMBBMBC
2h 0.4980.4760.4570.4480.4850.60.3450.3510.371
2.5h 0.7230.640.6450.7090.6880.5540.6390.6560.658
GFP fluorescenceMLAMLBMLCMSAMSBMSCMBAMBBMBC
2h 8208492717585971148478952-2-46-16
2.5h 1556771906937776271696212-142495

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
0.8mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD600 1.6461.5761.6431.7021.8321.8262.2722.0901.985
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.

OD600MLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 0.5410.590.5620.6160.5960.5620.4860.4980.46
3.5h 0.8030.820.8170.9210.8720.840.8060.8110.796
GFP fluorescenceMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 133519133447117828115401125899112488859842773
3.5h 178972208432157171160958151790145594181315631595
RPUML-AML-BML-CaveragestdevCV
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
2mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD600 3.0673.0673.0672.9362.9362.9362.3222.3222.322
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.

OD600MLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 0.420.4370.4170.4750.4380.4840.4380.4080.419
3.5h0.5740.6280.5930.6520.6440.7070.6820.6310.653
GFP fluorescenceMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 888578222386475865457667785028758661695
3.5h 12431612897612015410887310782811308211039811061
RPUML-AML-BML-CaveragestdevCV
2mM1.48991.8341.39081.57160.23260.148


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
0.3mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD600 3.0993.0993.0993.143.143.143.1813.1813.181
0.03mMML-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD600 3.1923.1923.1923.183.183.183.2473.2473.247
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 0.3mMMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 0.50.4920.5020.5430.4990.5220.6340.6150.63
4h 0.830.850.8780.9120.8550.8931.3271.2771.318
GFP fluorescence IPTG 0.3mMMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 1053419485910157110690594079110943111510391007
4h 153111143951152096170446149843153449201318511883
RPUML-AML-BML-CaveragestdevCV
0.3mM 0.93780.95450.95510.94910.00980.0103
OD600 IPTG 0.03mMMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 0.5050.4990.5170.5190.5010.5280.6230.6510.631
4h 0.9030.8610.9090.8890.8810.9091.2761.3521.319
GFP fluorescence IPTG 0.03mMMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 331372800034213101336917718877810571173999
4h 476914037049962190889167666163445189920811976
RPUML-AML-BML-CaveragestdevCV
0.03mM 0.176610.155110.189350.173690.017310.09966


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-AML-BML-CMS-AMS-BMS-CMB-AMB-BMB-C
OD6003.1533.1533.1532.9052.9052.9053.183.183.18
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 0.3mMMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 0.5490.5460.5420.510.5190.5120.8470.813X
3.5h 0.7230.7260.7180.6780.6680.6671.2261.1911.195
GFP fluorescence IPTG 0.3mMMLAMLBMLCMSAMSBMSCMBAMBBMBC
3h 331372800034213101336917718877810571173999
3.5h 476914037049962190889167666163445189920811976
RPUML-AML-BML-CaveragestdevCV
0.03mM 0.1650.13930.18060.16160.02090.1291