Team:Kyoto/Notebook3

From 2010.igem.org

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(→Monday, October 4 By: Ken)
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We couldn't observed the cell lysis.
We couldn't observed the cell lysis.
 +
====Tuesday, October 8 <span class="by">By: Ken</span>====
====Tuesday, October 8 <span class="by">By: Ken</span>====
Line 102: Line 103:
====Sunday, October 10 <span class="by">By: Ken</span>====
====Sunday, October 10 <span class="by">By: Ken</span>====
-
We cultivated E.coli transformed with K350819, and extracted this plasmid, and id sequence analysis.
+
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.
As a result, R0011, a lactose promoter, had mutation as follows.
Line 108: Line 109:
We dicided to cultivated E.coli 30 degrees.
We dicided to cultivated E.coli 30 degrees.
-
----
+
====Saturday, October 16 <span class="by">By: Ken</span>====
 +
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.
 +
{| class="experiments"
 +
!||0||75||145||235||290||350||390||450
 +
|-
 +
|0mM(-)||0.138||0.228||0.349||0.513||0.674||0.899||1.125||1.46
 +
|-
 +
|0.1mM(-)||0.138||0.227||0.347||0.515||0.667||0.886||1.025||1.369
 +
|-
 +
|1.0mM(-)||0.138||0.222||0.341||0.346||0.181||0.085||0.091||0.078
 +
|}
 +
{| class="experiments"
 +
!||0||60||100||170||210||270
 +
|-
 +
|0mM(+)||0.154||0.296||0.379||0.622||0.985||1.566
 +
|-
 +
|1mM(+)||0.154||0.243||0.365||0.609||0.89||0.608
 +
|}
 +
The growth with M9 wasn't enough to observe the cell death. So, we decided to choose M9+casamino acids.
 +
 
 +
====Sunday, October 17 <span class="by">By: Ken</span>====
 +
=====Measurement of lysis cassette=====
 +
According to those experiments already shown, we checked the function of lac-SRRz and if the cultural condition were suitable for observing the cell death.
 +
So, we set the cultural temperature to 30 degrees and used M9+casamino acids.
 +
{| class="experiments"
 +
!||0||75||155||240||285||345||420||500||560
 +
|-
 +
|0mM||0.112||0.191||0.383||0.763||1.071||1.637||2.471||2.935||2.901
 +
|-
 +
|0.01mM||0.112||0.187||0.369||0.742||||1.602||2.429||2.788||2.905
 +
|-
 +
|0.02mM||0.112||0.18||0.371||0.742||||1.575||2.403||2.713||2.777
 +
|-
 +
|0.03mM||0.112||0.175||0.357||0.735||||1.532||2.332||2.697||2.731
 +
|-
 +
|1.0mM||0.112||0.187||0.374||0.511||0.316||0.191||0.187||0.203||0.222
 +
|}
 +
=====Measurement of lysisbox-module2-=====
 +
We also measured lysisbox-module2- with the same cultural condition.
 +
{| class="experiments"
 +
!|||0||75||150||225||270||345||420||510||570
 +
|-
 +
|0mM||0.168||0.316||0.604||1.077||1.545||2.271||2.657||2.853||2.913
 +
|-
 +
|0.03mM||0.168||0.297||0.560||1.015||||2.150||2.601||2.333||2.075
 +
|-
 +
|0.1mM||0.168||0.310||0.598||1.060||||2.241||1.778||0.463||0.500
 +
|-
 +
|1.0mM||0.168||0.308||0.580||0.807||0.701||0.536||0.424|||
 +
|}
 +
 
 +
 
 +
 
 +
====Wednesday, October 20 <span class="by">By: Ken</span>====
 +
=====Measurement of lac-SRRz=====
 +
{| class="experiments"
 +
!||0||120||180||280||340||460
 +
|-
 +
|0mM||0.110||0.467||0.867||1.630||1.985||2.358
 +
|-
 +
|0.01mM||0.110||0.445||0.847||1.598||1.895||2.297
 +
|-
 +
|0.02mM||0.110||0.447||0.815||1.527||1.848||2.430
 +
|-
 +
|0.03mM||0.110||0.439||0.803||1.558||1.891||2.028
 +
|-
 +
|0.1mM||0.110||0.457||0.843||1.620||1.851||0.269
 +
|-
 +
|0.5mM||0.110||0.424||0.793||0.383||0.123||0.123
 +
|-
 +
|1.0mM||0.110||0.452||0.693||0.138||0.113||0.102
 +
|-
 +
|2.0mM||0.110||0.422||0.292||0.059||0.056||0.066
 +
|}
 +
=====Measurement of lysisbox-module2-=====
 +
{| class="experiments"
 +
!||0||120||180||280||340||460
 +
|-
 +
|0mM||0.109||0.363||0.659||1.610||1.923||2.619
 +
|-
 +
|0.02mM||0.109||0.355||0.641||1.509||1.916||2.465
 +
|-
 +
|0.03mM||0.109||0.341||0.633||1.469||1.898||1.756
 +
|-
 +
|0.05mM||0.109||0.343||0.613||1.470||1.805||1.231
 +
|-
 +
|0.3mM||0.109||0.363||0.622||1.369||0.409||0.476
 +
|-
 +
|0.5mM||0.109||0.315||0.580||1.004||0.208||0.429
 +
|-
 +
|0.7mM||0.109||0.353||0.494||0.559||0.447||0.314
 +
|-
 +
|1.0mM||0.109||||0.434||0.310||0.268||0.542
 +
|}
 +
 
 +
 
 +
 
 +
====Tuesday, October 26 <span class="by">By: Ken</span>====
 +
=====Measurement of lac-SRRz=====
 +
{|
 +
!Incubating
 +
|16h||18h||20h||16h||18h||20h||16h||18h||20h
 +
|-
 +
!Number of colony
 +
|1||1||1||2||2||2||3||3||3
 +
|-
 +
!0mM
 +
|2.13||2.17||2.35||2.05||2.04||2.14||2.22||2.21||2.33
 +
|-
 +
!0.01mM
 +
|2.02||2.01||2.15||1.96||2.23||2.23||2.21||2.07||2.29
 +
|-
 +
!0.02mM
 +
|2.21||2.12||2.16||1.86||1.86||1.95||1.76||1.93||1.71
 +
|-
 +
!0.03mM
 +
|1.44||1.41||1.39||1.70||1.62||1.59||0.82||1.02||0.83
 +
|-
 +
!0.05mM
 +
|0.33||0.25||0.17||0.25||0.25||0.24||0.26||0.33||0.24
 +
|-
 +
!0.07mM
 +
|0.15||0.17||0.23||0.21||0.20||0.23||0.18||0.18||0.19
 +
|-
 +
!0.1mM
 +
|0.13||0.20||0.36||0.20||0.18||0.23||0.17||0.15||0.23
 +
|-
 +
!0.3mM
 +
|0.10||0.21||0.31||0.08||0.11||0.14||0.11||0.07||0.20
 +
|-
 +
!0.5mM
 +
|0.07||0.08||0.10||0.08||0.08||0.09||0.11||0.08||0.25
 +
|-
 +
!0.8mM
 +
|0.25||0.10||0.08||0.29||0.22||0.11||0.18||0.22||0.10
 +
|-
 +
!1.0mM
 +
|0.04||0.15||0.21||0.18||0.27||0.27||0.18||0.06||0.24
 +
|}
 +
 
 +
=====Measurement of lac-S<sub>&Delta;TMD1</sub>RRz=====
 +
{|
 +
!Incubating
 +
|16h||18h||20h||16h||18h||20h
 +
|-
 +
!Number of colony
 +
|1||1||1||2||2||2
 +
|-
 +
!0mM
 +
|1.149||1.692||2.352||2.027||2.485||2.559
 +
|-
 +
!1mM
 +
|1.350||1.971||2.356||1.682||2.177||2.706
 +
|}
 +
 
 +
====Wednesday, October 27 <span class="by">By: Ken</span>====
 +
=====Measurement of lac-SRRz=====
 +
{| class="experiments"
 +
!||0h||0.5h||1.0h||1.5h||2.0h||2.5h||3.0h||3.5h||4.0h||4.5h||5.0h||5.5h||6.0h||6.5h||7.0h
 +
|-
 +
|0mM||0.169||0.277||0.392||0.559||0.751||0.978||1.177||1.375||1.634||1.813||1.998||2.097||2.115||2.200||2.205
 +
|-
 +
|0.03mM||0.170||0.274||0.391||0.550||0.722||0.974||1.175||1.384||1.654||1.892||2.002||2.034||1.981||1.681||1.412
 +
|-
 +
|0.05mM||0.158||0.258||0.367||0.512||0.689||0.911||1.122||1.350||1.524||1.679||1.522||1.004||0.732||0.503||0.387
 +
|-
 +
|0.1mM||0.167||0.273||0.388||0.562||0.768||1.028||1.218||0.700||0.749||0.518||0.210||0.189||0.195||0.190||0.198
 +
|-
 +
|0.3mM||0.164||0.267||0.392||0.552||0.727||0.896||0.685||0.269||0.118||0.110||0.112||0.118||0.124||0.131||0.135
 +
|-
 +
|0.5mM||0.159||0.254||0.364||0.483||0.542||0.283||0.115||0.078||0.080||0.075||0.076||0.075||0.074||0.082||0.087
 +
|-
 +
|1.0mM||0.156||0.255||0.357||0.411||0.314||0.093||0.069||0.059||0.053||0.052||0.049||0.049||0.050||0.049||0.057
 +
|}
 +
 
 +
=====Measurement of lac-S<sub>&Delta;TMD1</sub>RRz=====
 +
{|
 +
!Incubating
 +
|16h||18h||20h||16h||18h||20h||16h||18h||20h||16h||18h||20h
 +
|-
 +
!Number of colony
 +
|1||1||1||2||2||2||3||3||3||4||4||4
 +
|-
 +
!0mM
 +
|2.699||2.769||2.908||2.619||2.770||2.870||2.669||2.789||2.922||2.798||2.908||3.045
 +
|-
 +
!1.0mM
 +
|2.644||2.657||2.720||2.677||2.886||2.948||2.688||2.817||2.811||2.583||2.787||2.697
 +
|}

Latest revision as of 21:42, 27 October 2010

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.

Sunday, October 17 By: Ken

Measurement of lysis cassette

According to those experiments already shown, we checked the function of lac-SRRz and if the cultural condition were suitable for observing the cell death. So, we set the cultural temperature to 30 degrees and used M9+casamino acids.

075155240285345420500560
0mM0.1120.1910.3830.7631.0711.6372.4712.9352.901
0.01mM0.1120.1870.3690.7421.6022.4292.7882.905
0.02mM0.1120.180.3710.7421.5752.4032.7132.777
0.03mM0.1120.1750.3570.7351.5322.3322.6972.731
1.0mM0.1120.1870.3740.5110.3160.1910.1870.2030.222
Measurement of lysisbox-module2-

We also measured lysisbox-module2- with the same cultural condition.

075150225270345420510570
0mM0.1680.3160.6041.0771.5452.2712.6572.8532.913
0.03mM0.1680.2970.5601.0152.1502.6012.3332.075
0.1mM0.1680.3100.5981.0602.2411.7780.4630.500
1.0mM0.1680.3080.5800.8070.7010.5360.424


Wednesday, October 20 By: Ken

Measurement of lac-SRRz
0120180280340460
0mM0.1100.4670.8671.6301.9852.358
0.01mM0.1100.4450.8471.5981.8952.297
0.02mM0.1100.4470.8151.5271.8482.430
0.03mM0.1100.4390.8031.5581.8912.028
0.1mM0.1100.4570.8431.6201.8510.269
0.5mM0.1100.4240.7930.3830.1230.123
1.0mM0.1100.4520.6930.1380.1130.102
2.0mM0.1100.4220.2920.0590.0560.066
Measurement of lysisbox-module2-
0120180280340460
0mM0.1090.3630.6591.6101.9232.619
0.02mM0.1090.3550.6411.5091.9162.465
0.03mM0.1090.3410.6331.4691.8981.756
0.05mM0.1090.3430.6131.4701.8051.231
0.3mM0.1090.3630.6221.3690.4090.476
0.5mM0.1090.3150.5801.0040.2080.429
0.7mM0.1090.3530.4940.5590.4470.314
1.0mM0.1090.4340.3100.2680.542


Tuesday, October 26 By: Ken

Measurement of lac-SRRz
Incubating 16h18h20h16h18h20h16h18h20h
Number of colony 111222333
0mM 2.132.172.352.052.042.142.222.212.33
0.01mM 2.022.012.151.962.232.232.212.072.29
0.02mM 2.212.122.161.861.861.951.761.931.71
0.03mM 1.441.411.391.701.621.590.821.020.83
0.05mM 0.330.250.170.250.250.240.260.330.24
0.07mM 0.150.170.230.210.200.230.180.180.19
0.1mM 0.130.200.360.200.180.230.170.150.23
0.3mM 0.100.210.310.080.110.140.110.070.20
0.5mM 0.070.080.100.080.080.090.110.080.25
0.8mM 0.250.100.080.290.220.110.180.220.10
1.0mM 0.040.150.210.180.270.270.180.060.24
Measurement of lac-SΔTMD1RRz
Incubating 16h18h20h16h18h20h
Number of colony 111222
0mM 1.1491.6922.3522.0272.4852.559
1mM 1.3501.9712.3561.6822.1772.706

Wednesday, October 27 By: Ken

Measurement of lac-SRRz
0h0.5h1.0h1.5h2.0h2.5h3.0h3.5h4.0h4.5h5.0h5.5h6.0h6.5h7.0h
0mM0.1690.2770.3920.5590.7510.9781.1771.3751.6341.8131.9982.0972.1152.2002.205
0.03mM0.1700.2740.3910.5500.7220.9741.1751.3841.6541.8922.0022.0341.9811.6811.412
0.05mM0.1580.2580.3670.5120.6890.9111.1221.3501.5241.6791.5221.0040.7320.5030.387
0.1mM0.1670.2730.3880.5620.7681.0281.2180.7000.7490.5180.2100.1890.1950.1900.198
0.3mM0.1640.2670.3920.5520.7270.8960.6850.2690.1180.1100.1120.1180.1240.1310.135
0.5mM0.1590.2540.3640.4830.5420.2830.1150.0780.0800.0750.0760.0750.0740.0820.087
1.0mM0.1560.2550.3570.4110.3140.0930.0690.0590.0530.0520.0490.0490.0500.0490.057
Measurement of lac-SΔTMD1RRz
Incubating 16h18h20h16h18h20h16h18h20h16h18h20h
Number of colony 111222333444
0mM 2.6992.7692.9082.6192.7702.8702.6692.7892.9222.7982.9083.045
1.0mM 2.6442.6572.7202.6772.8862.9482.6882.8172.8112.5832.7872.697
Retrieved from "http://2010.igem.org/Team:Kyoto/Notebook3"