Team:Kyoto/Notebook3

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.

	0	30	60	90	120	150	180	210	240
0mM	0.22	0.38	0.57	0.89	1.20	1.52	1.80	2.00	2.03
0.1mM	0.22	0.41	0.57	0.85	1.14	0.71	0.40	0.13	0.17
0.2mM	0.22	0.40	0.55	0.86	0.26	0.11	0.09	0.10	0.11
0.3mM	0.22	0.39	0.54	0.79	0.16	0.08	0.09	0.10	0.10
0.4mM	0.22	0.37	0.49	0.64	0.05	0.02	0.03	0.04	0.06
0.5mM	0.22	0.40	0.54	0.64	0.08	0.06	0.07	0.08	0.08

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.

	0	30	60	90	120	150	180	210	240
0	0.36	0.56	0.97	1.36	1.69	1.84	1.94	1.99	2.00
0.01	0.22	0.51	0.74	1.12	1.50	1.76	1.91	1.92	1.92
0.03	0.26	0.56	0.75	1.12	1.51	1.76	1.37	0.91	0.68
0.05	0.27	0.58	0.76	1.11	1.49	1.56	0.50	0.24	0.25
0.07	0.25	0.52	0.73	1.12	1.39	1.08	0.18	0.17	0.16
0.1	0.26	0.55	0.75	1.09	1.07	0.77	0.14	0.17	0.17
1.0	0.26	0.55	0.77	0.13	0.12	0.13	0.18	0.15	0.12

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

	0	42	72	105	135	166	201	224
0mM	0.215	0.377	0.524	0.841	1.215	1.701	2.374	2.791
0.01mM	0.205	0.372	0.531	0.777	1.136	1.646	2.501	2.653
0.03mM	0.133	0.284	0.401	0.812	0.865	1.321	1.779	2.209
0.05mM	0.222	0.359	0.501	0.645	1.075	1.654	2.197	2.465
0.5mM	0.269	0.403	0.532	0.767	0.616	0.734	0.899	0.793

We couldn't observed the cell lysis.

Tuesday, October 8 By: Ken

	0	35	65	95	130	160	197	245	290
0mM	0.16	0.265	0.427	0.626	0.954	1.065	1.77	2.28	2.529
0.01mM	0.16	0.266	0.421	0.604	0.936	1.029	1.75	2.22	2.527
0.02mM	0.16	0.258	0.412	0.594	0.932	1	1.74	2.26	2.36
0.03mM	0.16	0.254	0.401	0.587	0.897	0.93	1.57	2.06	2.242
1.0mM	0.16	0.27	0.352	0.318	0.297	0.122	0.59	1	1.349

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.

	0	80	125	155	205
0mM	0.145	0.474	0.827	1.183	1.815
1mM	0.145	0.338	0.271	0.344	0.625
1mM(1/2)	0.145	0.389	0.34	0.366	0.646

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.

	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

	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 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.

	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.

	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 By: Ken

Measurement of lac-SRRz

	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-

	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 By: Ken

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_ΔTMD1RRz

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 By: Ken

Measurement of lac-SRRz

	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_ΔTMD1RRz

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