Our team participated in the Fifth International InterLaboratory Measurement Study this year. The interlab study aims to solve the problem of reliability and repeatability in synthetic biology study. This year we were asked to measure green fluorescent protein, which is used as a measurement marker. We followed the experiment protocol strictly to make sure our data is valid. After two weeks lab work, we got the data we need after several attempts.

Calibration Protocol

Three calibration measurement were done by the calibration protocol, including an OD reference point at 600nm, one particle standard curve and one fluorescent standard curve.

(Plate Reader: Molecular Device, Spectramax M5)

★ OD600 reference point ★

	LUDOX CL-X	H2O
Replicate 1	0.066	0.0285
Replicate 2	0.059	0.029
Replicate 3	0.068	0.026
Replicate 4	0.064	0.028
Arith. Mean	0.064	0.028
Corrected Abs600	0.036
Reference OD600	OD600
OD600/Abs600	1.728

Form1.Particle standard curve

Fluorescein_Standard_Curve_%28log_scale%29

Cell measurement

Transformation

Transform Escherichia coli DH5α with these following plasmids:

Device	Part Number	Location
Positive control	BBa_R0040	Well 2D
Negative control	Ba_I20270	Well 2B
Test Device 1	BBa_J364000	Well 2F
Test Device 2	BBa_J364001	Well 2H
Test Device 3	BBa_J364002	Well 2J
Test Device 4	BBa_J364007	Well 2L
Test Device 5	BBa_J364008	Well 2N
Test Device 6	BBa_J364009	Well 2P

The transformation used E.coli DH5α competent cells bought from Tsingke Biological Technology company, and follow the steps in http://parts.igem.org/Help:2017_DNA_Distribution to use the DNA in the Distribution Kit.

Measurement

For this part of inter lab study, we followed the following protocols:

1.Grown 8 devices in incubator for 12 hrs at 37 ℃
2.Pick 2 colonies from each of plate and inocubate them on 10mL LB medium with Chloramphenicol. Grow the cells for 16-20hrs at 37°C and 180rpm.
3.Measure OD600nm of the overnight cultures and record the data, then dilute to target OD600nm = 0.02 in the falcon tubes.
4.Measure the OD600nm and Fl under the same condition as standard curve measurement and use the same 96 wells plates.

★ Fluorescence Raw Readings：★

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	98.891	514.509	170.45	453.211	107.527	377.998	110.335	121.276	100.56
Colony 1, Replicate 2	87.951	520.569	155.638	443.722	103.159	353.38	99.569	117.851	97.983
Colony 1, Replicate 3	89.665	500.61	151.21	474.651	99.334	358.843	97.329	115.674	97.686
Colony 1, Replicate 4	88.651	553.489	158.106	471.047	107.222	391.116	102.558	132.724	104.901
Colony 2, Replicate 1	94.201	799.161	176.469	396.183	105.65	452.456	105.862	129.379	100.254
Colony 2, Replicate 2	97.455	750.414	168.71	425.555	106.286	462.598	109.024	125.311	101.914
Colony 2, Replicate 3	95.836	795.354	174.674	405.952	103.704	444.673	106.365	128.606	105.761
Colony 2, Replicate 4	100.733	804.913	203.631	370.775	117.227	506.476	113.511	125.021	105.18

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	142.426	1579.487	407.651	2268.46	167.703	1014.837	144.06	365.966	130.779
Colony 1, Replicate 2	143.238	1593.226	396.943	2328.22	174.862	1003.434	141.231	339.499	126.638
Colony 1, Replicate 3	151.515	1699.317	396.669	2175.501	169.71	977.6	143.762	347.711	127.911
Colony 1, Replicate 4	155.799	1783.066	406.205	2240.128	171.295	1044.087	154.138	340.887	127.237
Colony 2, Replicate 1	150.46	2062.959	354.432	2090.23	165.189	1268.176	150.674	320.936	120.878
Colony 2, Replicate 2	145.579	2023.17	366.367	2169.758	167.611	1203.787	144.458	322.957	127.103
Colony 2, Replicate 3	146.553	2101.747	353.019	1926.808	183.724	1172.553	158.135	332.951	131.201
Colony 2, Replicate 4	157.503	2071.459	316.402	1750.215	179.275	1253.449	155.625	327.385	137.828

★ Abs600 Raw Readings：★

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0.1306	0.0884	0.0767	0.1068	0.1181	0.0753	0.1067	0.0866	0.0486
Colony 1, Replicate 2	0.1272	0.0906	0.0737	0.1075	0.1184	0.0754	0.108	0.0896	0.0497
Colony 1, Replicate 3	0.1246	0.09	0.0747	0.1119	0.1214	0.0731	0.1083	0.0879	0.0464
Colony 1, Replicate 4	0.1225	0.0872	0.0743	0.1109	0.1247	0.0746	0.1079	0.0903	0.0476
Colony 2, Replicate 1	0.1301	0.0907	0.0701	0.1109	0.1132	0.0813	0.1103	0.0909	0.0468
Colony 2, Replicate 2	0.1327	0.0919	0.0685	0.1125	0.1145	0.0822	0.1081	0.0896	0.0472
Colony 2, Replicate 3	0.1294	0.0952	0.0693	0.1151	0.1139	0.0809	0.1084	0.0898	0.05
Colony 2, Replicate 4	0.1202	0.0941	0.0692	0.1105	0.1134	0.0805	0.1092	0.089	0.0472

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0.9386	0.7564	0.1978	0.911	0.7963	0.2566	0.8131	0.6363	0.0444
Colony 1, Replicate 2	0.9413	0.7528	0.1974	0.9012	0.8211	0.2547	0.7951	0.5996	0.0438
Colony 1, Replicate 3	0.9745	0.7748	0.1951	0.8642	0.81	0.2536	0.7994	0.6044	0.0458
Colony 1, Replicate 4	0.9932	0.7975	0.2009	0.8969	0.8021	0.2622	0.8266	0.6092	0.0438
Colony 2, Replicate 1	0.8246	0.78	0.1031	0.8625	0.8193	0.2834	0.7633	0.5251	0.0457
Colony 2, Replicate 2	0.8327	0.7665	0.1031	0.9018	0.8211	0.2781	0.7394	0.5374	0.0462
Colony 2, Replicate 3	0.8062	0.7927	0.1005	0.829	0.8082	0.276	0.7689	0.5668	0.0458
Colony 2, Replicate 4	0.8262	0.7805	0.1005	0.8787	0.8156	0.2928	0.7604	0.5332	0.0477

★ uM Fluorescein / OD ★

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
olony 1, Replicate 1	-0.002	0.872	0.208	0.508	0.008	0.871	0.014	0.046
Colony 1, Replicate 2	-0.011	0.866	0.201	0.501	0.006	0.833	0.002	0.042
Colony 1, Replicate 3	-0.009	0.775	0.159	0.482	0.002	0.820	0.000	0.036
Colony 1, Replicate 4	-0.018	0.950	0.167	0.485	0.003	0.889	-0.003	0.055
Colony 2, Replicate 1	-0.006	1.334	0.274	0.387	0.007	0.856	0.007	0.055
Colony 2, Replicate 2	-0.004	1.216	0.263	0.415	0.005	0.864	0.010	0.046
Colony 2, Replicate 3	-0.010	1.279	0.299	0.387	-0.003	0.919	0.001	0.048
Colony 2, Replicate 4	-0.005	1.251	0.375	0.352	0.015	1.010	0.011	0.040

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.001	0.171	0.151	0.207	0.004	0.349	0.001	0.033
Colony 1, Replicate 2	0.002	0.173	0.148	0.215	0.005	0.348	0.002	0.032
Colony 1, Replicate 3	0.002	0.181	0.151	0.210	0.005	0.343	0.002	0.033
Colony 1, Replicate 4	0.003	0.184	0.149	0.208	0.005	0.352	0.003	0.032
Colony 2, Replicate 1	0.003	0.222	0.341	0.202	0.005	0.405	0.003	0.035
Colony 2, Replicate 2	0.002	0.221	0.352	0.200	0.004	0.389	0.002	0.033
Colony 2, Replicate 3	0.002	0.221	0.340	0.192	0.006	0.379	0.003	0.032
Colony 2, Replicate 4	0.002	0.221	0.283	0.163	0.005	0.382	0.002	0.033

★ Net Fluorescein a.u. ★

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	-1.67	413.95	69.89	352.65	6.97	277.44	9.77	20.72
Colony 1, Replicate 2	-10.03	422.59	57.66	345.74	5.18	255.40	1.59	19.87
Colony 1, Replicate 3	-8.02	402.92	53.52	376.97	1.65	261.16	-0.36	17.99
Colony 1, Replicate 4	-16.25	448.59	53.21	366.15	2.32	286.22	-2.34	27.82
Colony 2, Replicate 1	-6.05	698.91	76.22	295.93	5.40	352.20	5.61	29.13
Colony 2, Replicate 2	-4.46	648.50	66.80	323.64	4.37	360.68	7.11	23.40
Colony 2, Replicate 3	-9.93	689.59	68.91	300.19	-2.06	338.91	0.60	22.85
Colony 2, Replicate 4	-4.45	699.73	98.45	265.60	12.05	401.30	8.33	19.84

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	11.65	1448.71	276.87	2137.68	36.92	884.06	13.28	235.19
Colony 1, Replicate 2	16.60	1466.59	270.31	2201.58	48.22	876.80	14.59	212.86
Colony 1, Replicate 3	23.60	1571.41	268.76	2047.59	41.80	849.69	15.85	219.80
Colony 1, Replicate 4	28.56	1655.83	278.97	2112.89	44.06	916.85	26.90	213.65
Colony 2, Replicate 1	29.58	1942.08	233.55	1969.35	44.31	1147.30	29.80	200.06
Colony 2, Replicate 2	18.48	1896.07	239.26	2042.66	40.51	1076.68	17.36	195.85
Colony 2, Replicate 3	15.35	1970.55	221.82	1795.61	52.52	1041.35	26.93	201.75
Colony 2, Replicate 4	19.68	1933.63	178.57	1612.39	41.45	1115.62	17.80	189.56

★ Net Abs 600 ★

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.082	0.040	0.028	0.058	0.070	0.027	0.058	0.038
Colony 1, Replicate 2	0.078	0.041	0.024	0.058	0.069	0.026	0.058	0.040
Colony 1, Replicate 3	0.078	0.044	0.028	0.066	0.075	0.027	0.062	0.042
Colony 1, Replicate 4	0.075	0.040	0.027	0.063	0.077	0.027	0.060	0.043
Colony 2, Replicate 1	0.083	0.044	0.023	0.064	0.066	0.035	0.064	0.044
Colony 2, Replicate 2	0.086	0.045	0.021	0.065	0.067	0.035	0.061	0.042
Colony 2, Replicate 3	0.079	0.045	0.019	0.065	0.064	0.031	0.058	0.040
Colony 2, Replicate 4	0.073	0.047	0.022	0.063	0.066	0.033	0.062	0.042

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.894	0.712	0.153	0.867	0.752	0.212	0.769	0.592
Colony 1, Replicate 2	0.898	0.709	0.154	0.8576	0.777	0.211	0.751	0.55
Colony 1, Replicate 3	0.929	0.729	0.149	0.818	0.764	0.208	0.754	0.559
Colony 1, Replicate 4	0.949	0.754	0.157	0.853	0.758	0.218	0.783	0.565
Colony 2, Replicate 1	0.779	0.734	0.057	0.817	0.774	0.238	0.718	0.479
Colony 2, Replicate 2	0.787	0.720	0.057	0.856	0.775	0.232	0.693	0.491
Colony 2, Replicate 3	0.760	0.747	0.055	0.783	0.762	0.230	0.723	0.521
Colony 2, Replicate 4	0.779	0.733	0.053	0.831	0.768	0.245	0.713	0.486

CFU Protocol

For this part of inter lab study, we followed the following protocol:

1.Pick two colonies from positive control device and negative control device, incubate overnight.
2.Prepare Starting Samples: Dilute the overnight cultures 1:8, measurement then dilute further to OD600nm = 0.1
Calculation:
Use (C1)(V1) = (C2)(V2) to calculate your dilutions C1 is your starting OD600 C2 is your target OD600 of 0.1
V1 is the unknown volume in μL V2 is the final volume of 1000 μL
3.Check and make sure OD600 = 0.1
4.Dilute Dilute
5.Incubate at 37 degree Celsius overnight for 18-20 hrs. Then count colony number.
colony_number

Team:ZJUT-China/InterLab

INTERLAB