Team:Kyoto/Notebook3

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Contents

Notebook3

Measurement of Lysis Cassette

Sunday, September 26 By: Wataru, Ken

After overnight pre-culture, dilute with supplemented M9 media as OD550 was 0.22, and measure OD550 every 30min. At the point of 30min, we add IPTG and continued to measure OD550. We used test tubes to cultivate E.coli. We cultivated E.coli in test tubes at 37 degreees.

0306090120150180210240
0mM0.220.380.570.891.201.521.802.002.03
0.1mM0.220.410.570.851.140.710.400.130.17
0.2mM0.220.400.550.860.260.110.090.100.11
0.3mM0.220.390.540.790.160.080.090.100.10
0.4mM0.220.370.490.640.050.020.030.040.06
0.5mM0.220.400.540.640.080.060.070.080.08

Discussion:

We can observe the cell lysis and this result suggest that the timing of the start of cell lysis is depending on the concentration of IPTG.

0306090120150180210240
00.360.560.971.361.691.841.941.992.00
0.010.220.510.741.121.501.761.911.921.92
0.030.260.560.751.121.511.761.370.910.68
0.050.270.580.761.111.491.560.500.240.25
0.070.250.520.731.121.391.080.180.170.16
0.10.260.550.751.091.070.770.140.170.17
1.00.260.550.770.130.120.130.180.150.12

Discussion:

When the concentration of IPTG is over 0.03mM, cell lysis occurs. We continued to incubate these cultures overnight.


Monday, September 27 By: Wataru, Ken

All of the cultures was over 2.0 of OD550. We caouln't find out the cause of it.


Monday, October 4 By: Ken

04272105135166201224
0mM0.2150.3770.5240.8411.2151.7012.3742.791
0.01mM0.2050.3720.5310.7771.1361.6462.5012.653
0.03mM0.1330.2840.4010.8120.8651.3211.7792.209
0.05mM0.2220.3590.5010.6451.0751.6542.1972.465
0.5mM0.2690.4030.5320.7670.6160.7340.8990.793

We couldn't observed the cell lysis.


Tuesday, October 8 By: Ken

0356595130160197245290
0mM0.160.2650.4270.6260.9541.0651.772.282.529
0.01mM0.160.2660.4210.6040.9361.0291.752.222.527
0.02mM0.160.2580.4120.5940.93211.742.262.36
0.03mM0.160.2540.4010.5870.8970.931.572.062.242
1.0mM0.160.270.3520.3180.2970.1220.5911.349

In 1mM IPTG, the cell lysis surely occured, however, E.coli started to grow soony.


Saturday, October 9 By: Ken

We considered that the cause of these unclear result was a degration of antibody. So we added kanamysin to be 100ug/ml and 50ug/ml and measured the difference.

080125155205
0mM0.1450.4740.8271.1831.815
1mM0.1450.3380.2710.3440.625
1mM(1/2)0.1450.3890.340.3660.646

As a result, the amount of kanamysin is not related.

Sunday, October 10 By: Ken

We cultivated E.coli transformed with K358019, and extracted this plasmid, and did sequence analysis. As a result, R0011, a lactose promoter, had mutation as follows.

This is because K350819 is over 7000bp and easier to have turn over and λ lysis cassette is toxic. We dicided to cultivated E.coli 30 degrees.

Saturday, October 16 By: Ken

We did the measurement of cell lysis with M9 or M9+casamino acids, and observed the differences. The table upper is the measurement with M9 and below is the measurement with M9+casamino acids.

075145235290350390450
0mM(-)0.1380.2280.3490.5130.6740.8991.1251.46
0.1mM(-)0.1380.2270.3470.5150.6670.8861.0251.369
1.0mM(-)0.1380.2220.3410.3460.1810.0850.0910.078
060100170210270
0mM(+)0.1540.2960.3790.6220.9851.566
1mM(+)0.1540.2430.3650.6090.890.608

The growth with M9 wasn't enough to observe the cell death. So, we decided to choose M9+casamino acids.