Difference between revisions of "Team:NCTU Formosa/InterLab"

This year, we contributed to the competition by participating in the 5th iGEM International Laboratory Study. As with previous years, the goal of the study is to standardize measurement techniques so that synthetic biology labs all over the world can reference and compare collected data reliably. This year’s focus is on converting fluorescence readings into absolute cell counts instead of relying on OD measurements, which can often vary from lab to lab. The materials required to perform this year’s interlab study include a plate reader capable of measuring fluorescence and competent E. coli DH5-alpha. We used a BMG labtech Clariostar plate reader along with black 96-well plates with transparent bottoms, and prepared DH5-alpha in glycerol stock. Using the rest of the required materials found in the iGEM interlab kit, we were able to test 8 plasmids consisting of a positive control, a negative control and 6 test constructs. We first performed calibrations to obtain standard values for our data.

Calibration 1 – LUDOX CL-X

Our first calibration uses LUDOX CL-X solution to convert absorbance (ABS₆₀₀) to an equivalent measurement of optical density (OD 600). This is necessary because absorbance measurements are dependent on the depth of the fluid sample, while OD 600 values obtained from a standard spectrophotometer depend only on cuvette width, which is constant. This calibration eliminates measurement variations specific to our plate reader and returns standard values of optical density that other labs can reference.

Table 1: Variable and Parameter used in temperature model.

	LUDOX CL-X	H₂O
Replicate 1	0.085	0.042
Replicate 2	0.088	0.063
Replicate 3	0.086	0.046
Replicate 4	0.085	0.054
Arith. Mean	0.086	0.051
Corrected Abs₆₀₀	0.035
Reference OD600	0.063
OD600/Abs₆₀₀	1.813

Calibration 2 – Microsphere Particle Standard Curve

For the next calibration, we prepared a dilution series of the silica microspheres found in the iGEM interlab kit. Because the concentration (particles per volume) is known, and because the microspheres have roughly the same size and optical qualities to bacterial cells, we can convert Abs₆₀₀ values to number of cells.

Table 2: Variable and Parameter used in temperature model.

Number of particles	2.35E+08	1.18E+08	5.88E+07	2.94E+07	1.47E+07	7.35E+06	3.68E+06	1.84E+06	9.19E+05	4.6E+05	2.3E+05	0
Replicate 1	0.884	0.536	0.277	0.231	0.087	0.057	0.045	0.036	0.036	0.032	0.030	0.029
Replicate 2	0.897	0.430	0.223	0.133	0.077	0.056	0.043	0.036	0.033	0.033	0.029	0.030
Replicate 3	1.082	0.417	0.226	0.124	0.075	0.054	0.061	0.039	0.037	0.067	0.033	0.029
Replicate 4	1.484	0.596	0.26	0.213	0.089	0.057	0.041	0.037	0.033	0.032	0.03	0.031
Arith. Mean	1.087	0.495	0.247	0.175	0.082	0.056	0.048	0.037	0.035	0.041	0.031	0.030
Arith. STDEV	0.280	0.086	0.026	0.055	0.007	0.001	0.009	0.001	0.002	0.017	0.002	0.001
Arith. Net Mean	1.057	0.465	0.217	0.146	0.052	0.026	0.018	0.007	0.005	0.011	0.001

Figure 1: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(A) Enterocin 96+intein+CBD(35.9kDa) (B) Enteroicin B+intein+CBD(35.5kDa)
(C)Bovicin HJ50+intein +CBD(34.25kDa) (D) Durancin +intein+CBD(35.3kDa)
(E) Lacticin+intein+CBD(33.9kDa) (F) Leucocyclicin Q+intein+CBD(34.4kDa)

Figure 2: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(A) Enterocin 96+intein+CBD(35.9kDa) (B) Enteroicin B+intein+CBD(35.5kDa)
(C)Bovicin HJ50+intein +CBD(34.25kDa) (D) Durancin +intein+CBD(35.3kDa)
(E) Lacticin+intein+CBD(33.9kDa) (F) Leucocyclicin Q+intein+CBD(34.4kDa)

Calibration 3 – Fluorescence Standard Curve

Our final calibration tackles the problem of inconsistencies in fluorescence measurements from model to model. In order to standardize our fluorescence values we create a standard fluorescence curve using the protein fluorescein. Measuring the fluorescence of a dilution series of fluorescein allows us to obtain a curve that describes the relationship between fluorescence values and fluorescein concentration.

Table 3: Variable and Parameter used in temperature model.

Fluorescein (uM)	10	5	2.5	1.25	0.625	0.313	0.156	0.078	0.039	0.0195	0.0098	0
Replicate 1	148873	85179	41074	23229	12491	5683	2437	1237	426	180	84	25
Replicate 2	151337	79470	41795	20500	10628	5217	2720	1378	684	368	173	22
Replicate 3	150211	84298	44059	22273	11429	5658	2892	1430	715	357	179	25
Replicate 4	153849	84629	45004	23125	11754	5867	2827	1548	890	425	235	21
Arith. Mean	1.511E+05	8.339E+04	4.298E+04	2.228E+04	1.158E+04	5.606E+03	2.719E+03	1.398E+03	6.788E+02	3.325E+02	1.678E+02	2.325E+01
Arith. STDEV	2.110E+03	2.641E+03	1.853E+03	1.263E+03	7.723E+02	2.757E+02	2.009E+02	1.289E+02	1.914E+02	1.059E+02	6.243E+01	2.062E+00
Arith. Net Mean	1.510E+05	8.337E+04	4.296E+04	2.226E+04	1.155E+04	5.583E+03	2.696E+03	1.375E+03	6.555E+02	3.093E+02	1.445E+02

Figure 3: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(A) Enterocin 96+intein+CBD(35.9kDa) (B) Enteroicin B+intein+CBD(35.5kDa)
(C)Bovicin HJ50+intein +CBD(34.25kDa) (D) Durancin +intein+CBD(35.3kDa)
(E) Lacticin+intein+CBD(33.9kDa) (F) Leucocyclicin Q+intein+CBD(34.4kDa)

Figure 4: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(A) Enterocin 96+intein+CBD(35.9kDa) (B) Enteroicin B+intein+CBD(35.5kDa)
(C)Bovicin HJ50+intein +CBD(34.25kDa) (D) Durancin +intein+CBD(35.3kDa)
(E) Lacticin+intein+CBD(33.9kDa) (F) Leucocyclicin Q+intein+CBD(34.4kDa)

Cell Measurement

Once our calibrations were complete, we moved on to the cell measurement protocol. First we transformed competent DH5-alpha with the 8 plasmids provided in the kit. After incubating for 16 hours at 37 degrees Celsius and 220 rpm, we diluted the solutions to an Abs₆₀₀ value of 0.02 before measuring both OD 600 as well as fluorescence at 0 hours and 6 hours. Then, using our previous calibrations we converted fluorescence per OD to fluorescence per particle.

Table 4: Fluorescence Raw Readings:

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + C^r (blank)
Colony 1, Replicate 1	906	1726	1167	777	1614	1026	1504	856	776
Colony 1, Replicate 2	792	1682	1285	827	1583	1328	1346	751	813
Colony 1, Replicate 3	886	1617	1403	910	1366	1141	763	766	762
Colony 1, Replicate 4	812	1515	1405	861	1525	1202	1220	794	789
Colony 2, Replicate 1	851	1661	1466	1012	1332	1035	1265	811	875
Colony 2, Replicate 2	773	1560	1569	1074	1296	1089	1192	771	922
Colony 2, Replicate 3	833	1590	1469	904	1333	1091	1295	768	862
Colony 2, Replicate 4	745	1695	1519	781	1278	812	1136	848	991

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + C^r (blank)
Colony 1, Replicate 1	975	5090	1471	1690	8573	1503	2042	942	894
Colony 1, Replicate 2	803	4603	1440	1601	8282	1476	1920	932	928
Colony 1, Replicate 3	926	4978	1427	1609	8246	1395	1847	882	1001
Colony 1, Replicate 4	850	4993	1566	1642	8688	1384	2113	1106	925
Colony 2, Replicate 1	1042	5576	1602	1171	4104	1276	1674	1063	940
Colony 2, Replicate 2	968	5312	1510	1188	4243	1368	1498	1111	893
Colony 2, Replicate 3	958	4621	1601	1343	4184	1419	1741	1136	937
Colony 2, Replicate 4	954	4957	1579	1095	3781	1255	1720	1036	984

Table 5: Fluorescence Raw Readings:

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + C^r (blank)
Colony 1, Replicate 1	0.089	0.088	0.083	0.078	0.066	0.068	0.07	0.227	0.034
Colony 1, Replicate 2	0.088	0.083	0.082	0.078	0.067	0.068	0.064	0.234	0.034
Colony 1, Replicate 3	0.087	0.08	0.082	0.08	0.061	0.066	0.036	0.065	0.035
Colony 1, Replicate 4	0.085	0.081	0.081	0.077	0.062	0.061	0.045	0.068	0.034
Colony 2, Replicate 1	0.08	0.091	0.079	0.082	0.07	0.073	0.073	0.298	0.036
Colony 2, Replicate 2	0.086	0.081	0.075	0.087	0.067	0.071	0.201	0.297	0.035
Colony 2, Replicate 3	0.082	0.085	0.074	0.082	0.07	0.069	0.075	0.257	0.033
Colony 2, Replicate 4	0.086	0.086	0.078	0.083	0.07	0.068	0.07	0.098	0.034

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + C^r (blank)
Colony 1, Replicate 1	0.762	0.459	0.467	0.497	0.78	0.632	0.652	0.539	0.038
Colony 1, Replicate 2	0.644	0.44	0.46	0.544	0.751	0.615	0.642	0.576	0.035
Colony 1, Replicate 3	0.653	0.475	0.558	0.538	0.85	0.613	0.661	0.589	0.04
Colony 1, Replicate 4	0.676	0.586	0.561	0.576	0.803	0.654	0.664	0.528	0.045
Colony 2, Replicate 1	0.559	0.507	0.476	0.457	0.498	0.743	0.687	0.655	0.036
Colony 2, Replicate 2	0.686	0.514	0.453	0.458	0.524	0.693	0.799	0.62	0.041
Colony 2, Replicate 3	0.655	0.553	0.512	0.519	0.607	0.889	0.86	0.648	0.037
Colony 2, Replicate 4	0.66	0.601	0.463	0.541	0.575	0.819	0.779	0.67	0.035

Table 6: uM Fluorescein / OD

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.074	0.552	0.250	0.001	0.822	0.231	0.634	0.013
Colony 1, Replicate 2	-0.012	0.556	0.309	0.010	0.732	0.475	0.557	-0.010
Colony 1, Replicate 3	0.075	0.596	0.428	0.103	0.729	0.384	0.031	0.004
Colony 1, Replicate 4	0.014	0.485	0.411	0.053	0.825	0.480	1.229	0.005
Colony 2, Replicate 1	-0.017	0.448	0.431	0.093	0.422	0.136	0.331	-0.008
Colony 2, Replicate 2	-0.092	0.435	0.507	0.092	0.367	0.146	0.051	-0.018
Colony 2, Replicate 3	-0.019	0.439	0.464	0.027	0.399	0.200	0.323	-0.013
Colony 2, Replicate 4	-0.148	0.425	0.376	-0.134	0.250	-0.165	0.126	-0.070

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.004	0.313	0.042	0.051	0.325	0.032	0.059	0.003
Colony 1, Replicate 2	-0.006	0.285	0.038	0.044	0.322	0.030	0.051	0.000
Colony 1, Replicate 3	-0.004	0.287	0.026	0.040	0.281	0.022	0.043	-0.007
Colony 1, Replicate 4	-0.004	0.236	0.039	0.049	0.321	0.024	0.060	0.012
Colony 2, Replicate 1	0.006	0.309	0.051	0.017	0.215	0.015	0.035	0.006
Colony 2, Replicate 2	0.004	0.293	0.044	0.022	0.218	0.023	0.025	0.012
Colony 2, Replicate 3	0.001	0.224	0.042	0.026	0.179	0.018	0.031	0.010
Colony 2, Replicate 4	-0.002	0.220	0.038	0.007	0.163	0.011	0.031	0.003

Table 7: Net Fluorescein a.u.

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	130.00	950.00	391.00	1.00	838.00	250.00	728.00	80.00
Colony 1, Replicate 2	-21.00	869.00	472.00	14.00	770.00	515.00	533.00	-62.00
Colony 1, Replicate 3	124.00	855.00	641.00	148.00	604.00	379.00	1.00	4.00
Colony 1, Replicate 4	23.00	726.00	616.00	72.00	736.00	413.00	431.00	5.00
Colony 2, Replicate 1	-24.00	786.00	591.00	137.00	457.00	160.00	390.00	-64.00
Colony 2, Replicate 2	-149.00	638.00	647.00	152.00	374.00	167.00	270.00	-151.00
Colony 2, Replicate 3	-29.00	728.00	607.00	42.00	471.00	229.00	433.00	-94.00
Colony 2, Replicate 4	-246.00	704.00	528.00	-210.00	287.00	-179.00	145.00	-143.00

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	81.00	4196.00	577.00	708.00	7679.00	609.00	1148.00	48.00
Colony 1, Replicate 2	-125.00	3675.00	512.00	582.00	7354.00	548.00	992.00	4.00
Colony 1, Replicate 3	-75.00	3977.00	426.00	600.00	7245.00	394.00	846.00	-119.00
Colony 1, Replicate 4	-75.00	4068.00	641.00	654.00	7763.00	459.00	1188.00	181.00
Colony 2, Replicate 1	102.00	4636.00	750.00	231.00	3164.00	336.00	734.00	123.00
Colony 2, Replicate 2	75.00	4419.00	708.00	295.00	3350.00	475.00	605.00	218.00
Colony 2, Replicate 3	21.00	3684.00	672.00	406.00	3247.00	482.00	804.00	199.00
Colony 2, Replicate 4	-30.00	3973.00	658.00	111.00	2797.00	271.00	736.00	52.00

Table 8: Variable and Parameter used in temperature model.

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.055	0.054	0.049	0.044	0.032	0.034	0.036	0.193
Colony 1, Replicate 2	0.054	0.049	0.048	0.044	0.033	0.034	0.030	0.200
Colony 1, Replicate 3	0.052	0.045	0.047	0.045	0.026	0.031	0.001	0.030
Colony 1, Replicate 4	0.051	0.047	0.047	0.043	0.028	0.027	0.011	0.034
Colony 2, Replicate 1	0.044	0.055	0.043	0.046	0.034	0.037	0.037	0.262
Colony 2, Replicate 2	0.051	0.046	0.040	0.052	0.032	0.036	0.166	0.262
Colony 2, Replicate 3	0.049	0.052	0.041	0.049	0.037	0.036	0.042	0.224
Colony 2, Replicate 4	0.052	0.052	0.044	0.049	0.036	0.034	0.036	0.064

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.724	0.421	0.429	0.438	0.742	0.594	0.614	0.501
Colony 1, Replicate 2	0.609	0.405	0.425	0.418	0.716	0.580	0.607	0.541
Colony 1, Replicate 3	0.613	0.435	0.518	0.472	0.810	0.573	0.621	0.549
Colony 1, Replicate 4	0.631	0.541	0.516	0.418	0.758	0.609	0.619	0.483
Colony 2, Replicate 1	0.523	0.471	0.461	0.421	0.462	0.707	0.651	0.619
Colony 2, Replicate 2	0.645	0.473	0.503	0.417	0.483	0.652	0.758	0.579
Colony 2, Replicate 3	0.618	0.516	0.501	0.482	0.570	0.852	0.823	0.611
Colony 2, Replicate 4	0.625	0.566	0.541	0.506	0.540	0.784	0.744	0.635

Table 9: Variable and Parameter used in temperature model.

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	2.34E+04	9.45E+03	2.71E+01	2.53E+04	1.04E+04	2.85E+04	2.95E+03	0.00E+00
Colony 1, Replicate 2	2.36E+04	1.16E+04	3.88E+02	2.33E+04	2.08E+04	2.08E+04	-2.75E+03	0.00E+00
Colony 1, Replicate 3	2.45E+04	1.57E+04	3.42E+03	1.70E+04	1.80E+04	-1.12E+03	-3.06E+04	-1.20E+03
Colony 1, Replicate 4	1.81E+04	1.38E+04	-3.96E+02	2.01E+04	1.55E+04	1.70E+04	-9.79E+03	-3.36E+03
Colony 2, Replicate 1	1.79E+04	1.64E+04	2.78E+03	1.19E+04	4.42E+03	1.23E+04	-3.99E+03	-2.39E+02
Colony 2, Replicate 2	2.02E+04	1.84E+04	7.05E+03	1.11E+04	9.43E+03	1.22E+04	-7.29E+02	3.04E+02
Colony 2, Replicate 3	1.53E+04	1.30E+04	-2.82E+03	9.28E+03	3.59E+03	1.12E+04	-7.06E+03	-7.66E+02
Colony 2, Replicate 4	1.80E+04	1.35E+04	-6.35E+03	7.79E+03	-6.61E+03	5.67E+03	-5.28E+03	0.00E+00

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	1.49E+02	1.33E+04	1.79E+03	2.15E+03	1.38E+04	1.36E+03	2.49E+03	1.27E+02
Colony 1, Replicate 2	-2.73E+02	1.21E+04	1.60E+03	1.85E+03	1.37E+04	1.26E+03	2.17E+03	9.83E+00
Colony 1, Replicate 3	-1.63E+02	1.22E+04	1.09E+03	1.69E+03	1.19E+04	9.14E+02	1.81E+03	-2.88E+02
Colony 1, Replicate 4	-1.58E+02	1.00E+04	1.65E+03	2.08E+03	1.36E+04	1.00E+03	2.55E+03	4.98E+02
Colony 2, Replicate 1	2.59E+02	1.31E+04	2.16E+03	7.30E+02	9.11E+03	6.32E+02	1.50E+03	2.64E+02
Colony 2, Replicate 2	1.55E+02	1.24E+04	1.87E+03	9.41E+02	9.22E+03	9.69E+02	1.06E+03	5.01E+02
Colony 2, Replicate 3	4.52E+01	9.49E+03	1.78E+03	1.12E+03	7.57E+03	7.52E+02	1.30E+03	4.33E+02
Colony 2, Replicate 4	-6.38E+01	9.33E+03	1.62E+03	2.92E+02	6.89E+03	4.60E+02	1.32E+03	1.09E+02

Table 10: Variable and Parameter used in temperature model.

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	950.00	391.00	1.00	838.00	250.00	728.00	80.00	0.00
Colony 1, Replicate 2	869.00	472.00	14.00	770.00	515.00	533.00	-62.00	0.00
Colony 1, Replicate 3	828.00	614.00	121.00	577.00	352.00	-26.00	-23.00	-27.00
Colony 1, Replicate 4	640.00	530.00	-14.00	650.00	327.00	345.00	-81.00	-86.00
Colony 2, Replicate 1	739.00	544.00	90.00	410.00	113.00	343.00	-111.00	-47.00
Colony 2, Replicate 2	698.00	707.00	212.00	434.00	227.00	330.00	-91.00	60.00
Colony 2, Replicate 3	599.00	478.00	-87.00	342.00	100.00	304.00	-223.00	-129.00
Colony 2, Replicate 4	704.00	528.00	-210.00	287.00	-179.00	145.00	-143.00	0.00

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	81.00	4196.00	577.00	708.00	7679.00	609.00	1148.00	48.00
Colony 1, Replicate 2	-125.00	3675.00	512.00	582.00	7354.00	548.00	992.00	4.00
Colony 1, Replicate 3	-75.00	3977.00	426.00	600.00	7245.00	394.00	846.00	-119.00
Colony 1, Replicate 4	-75.00	4068.00	641.00	654.00	7763.00	459.00	1188.00	181.00
Colony 2, Replicate 1	102.00	4636.00	750.00	231.00	3164.00	336.00	734.00	123.00
Colony 2, Replicate 2	75.00	4419.00	708.00	295.00	3350.00	475.00	605.00	218.00
Colony 2, Replicate 3	21.00	3684.00	672.00	406.00	3247.00	482.00	804.00	199.00
Colony 2, Replicate 4	-30.00	3973.00	658.00	111.00	2797.00	271.00	736.00	52.00

Table 11: Variable and Parameter used in temperature model.

Hour 0:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.054	0.055	0.049	0.044	0.032	0.034	0.036	0.193
Colony 1, Replicate 2	0.049	0.054	0.048	0.044	0.033	0.034	0.030	0.200
Colony 1, Replicate 3	0.045	0.052	0.047	0.045	0.026	0.031	0.001	0.030
Colony 1, Replicate 4	0.047	0.051	0.047	0.043	0.028	0.027	0.011	0.034
Colony 2, Replicate 1	0.055	0.044	0.043	0.046	0.034	0.037	0.037	0.262
Colony 2, Replicate 2	0.046	0.051	0.040	0.052	0.032	0.036	0.166	0.262
Colony 2, Replicate 3	0.052	0.049	0.041	0.049	0.037	0.036	0.042	0.224
Colony 2, Replicate 4	0.052	0.052	0.044	0.049	0.036	0.034	0.036	0.064

Hour 6:	N. Control	P. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	0.724	0.421	0.429	0.438	0.742	0.594	0.614	0.501
Colony 1, Replicate 2	0.609	0.405	0.425	0.418	0.716	0.580	0.607	0.541
Colony 1, Replicate 3	0.613	0.435	0.518	0.472	0.810	0.573	0.621	0.549
Colony 1, Replicate 4	0.631	0.541	0.516	0.418	0.758	0.609	0.619	0.483
Colony 2, Replicate 1	0.523	0.471	0.461	0.421	0.462	0.707	0.651	0.619
Colony 2, Replicate 2	0.645	0.473	0.503	0.417	0.483	0.652	0.758	0.579
Colony 2, Replicate 3	0.618	0.516	0.501	0.482	0.570	0.852	0.823	0.611
Colony 2, Replicate 4	0.625	0.566	0.541	0.506	0.540	0.784	0.744	0.635

Colony Formig Units

Finally, we test whether our cell measurement conversion was accurate. Because our conversions are based on the calibrations we performed using the silica beads, we observe actual colony forming units on plates and compare with our theoretical conversions. To do this, we incubated 2 samples of both positive and negative control overnight, then diluted 1 mL of the culture to an OD 600 value of 0.1 and prepared a series dilution. We then spread the final 3 dilutions of each sample onto plates and counted the CFUs. Because we started with an OD 600 value of 0.1, we know the OD values of the subsequent dilutions and can compare the resulting number of CFUs to our conversions from our cell measurement protocol.

Table 12: Variable and Parameter used in temperature model.
Positive Colony 1 Triplicate 1	1.8E+07 CFU/mL
Positive Colony 1 Triplicate 2	2.56E+07 CFU/mL
Positive Colony 1 Triplicate 3	5.6E+06 CFU/mL
Positive Colony 2 Triplicate 1	7.44E+07 CFU/mL
Positive Colony 2 Triplicate 2	1.04E+07 CFU/mL
Positive Colony 2 Triplicate 3	2.4E+06 CFU/mL
Negative Colony 1 Triplicate 1	2.23E+07 CFU/mL
Negative Colony 1 Triplicate 2	9.84E+07 CFU/mL
Negative Colony 1 Triplicate 3	1.6E+06 CFU/mL
Negative Colony 2 Triplicate 1	1.376E+08 CFU/mL
Negative Colony 2 Triplicate 2	1.008E+08 CFU/mL
Negative Colony 2 Triplicate 1	2.23E+07 CFU/mL

Table 13: Variable and Parameter used in temperature model.
Positive/Negative	Colony	Triplicate	Dilution	Data
Positive	Colony 1	Triplicate 1	Dilution 3	19
Positive	Colony 1	Triplicate 1	Dilution 4	23
Positive	Colony 1	Triplicate 1	Dilution 5	0
Positive	Colony 1	Triplicate 2	Dilution 3	44
Positive	Colony 1	Triplicate 2	Dilution 4	32
Positive	Colony 1	Triplicate 2	Dilution 5	0
Positive	Colony 1	Triplicate 3	Dilution 3	173
Positive	Colony 1	Triplicate 3	Dilution 4	7
Positive	Colony 1	Triplicate 3	Dilution 5	0
Positive	Colony 2	Triplicate 1	Dilution 3	20
Positive	Colony 2	Triplicate 1	Dilution 4	93
Positive	Colony 2	Triplicate 1	Dilution 5	2
Positive	Colony 2	Triplicate 2	Dilution 3	100
Positive	Colony 2	Triplicate 2	Dilution 4	13
Positive	Colony 2	Triplicate 2	Dilution 5	3
Positive	Colony 2	Triplicate 3	Dilution 3	3
Positive	Colony 2	Triplicate 3	Dilution 4	0
Positive	Colony 2	Triplicate 3	Dilution 5	0
Negative	Colony 1	Triplicate 1	Dilution 3	13
Negative	Colony 1	Triplicate 1	Dilution 4	29
Negative	Colony 1	Triplicate 1	Dilution 5	1368
Negative	Colony 1	Triplicate 2	Dilution 3	355
Negative	Colony 1	Triplicate 2	Dilution 4	123
Negative	Colony 1	Triplicate 2	Dilution 5	2
Negative	Colony 1	Triplicate 3	Dilution 3	431
Negative	Colony 1	Triplicate 3	Dilution 4	2
Negative	Colony 1	Triplicate 3	Dilution 5	1
Negative	Colony 2	Triplicate 1	Dilution 3	88
Negative	Colony 2	Triplicate 1	Dilution 4	172
Negative	Colony 2	Triplicate 1	Dilution 5	3
Negative	Colony 2	Triplicate 2	Dilution 3	370
Negative	Colony 2	Triplicate 2	Dilution 4	26
Negative	Colony 2	Triplicate 2	Dilution 5	1
Negative	Colony 2	Triplicate 3	Dilution 3	275
Negative	Colony 2	Triplicate 3	Dilution 4	29
Negative	Colony 2	Triplicate 3	Dilution 5	0

Extra credit – Flow Cytometry

As an extra credit task, we also collected flow cytometry data using an ACEA NovoCyte Flow Cytometer as well as SpheroTech Rainbow Calibration Particles model URCP-38-2K. Flow cytometry provides an even more accurate method of counting particles.

Conclusion

We hope our experimentation will help to improve the accessibility and comparability of reliable data in synthetic biology labs across the world. Thank you to iGEM for providing this opportunity to give back to the scientific community.

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      <p class="title_2">Colony Formig Units</p>
-     <div class="text"><p>Finally, we test whether our cell measurement conversion was accurate. Because our conversions are based on the calibrations we performed using the silica beads, we observe actual colony forming units on plates and compare with our theoretical conversions. To do this, we incubated 2 samples of both positive and negative control overnight, then diluted 1 mL of the culture to an OD 600 value of 0.1 and prepared a series dilution. We then spread the final 3 dilutions of each sample onto plates and counted the CFUs. Because we started with an OD 600 value of 0.1, we know the OD values of the subsequent dilutions and can compare the resulting number of CFUs to our conversions from our cell measurement protocol.</p></div>
+     <div class="text"><p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Finally, we test whether our cell measurement conversion was accurate. Because our conversions are based on the calibrations we performed using the silica beads, we observe actual colony forming units on plates and compare with our theoretical conversions. To do this, we incubated 2 samples of both positive and negative control overnight, then diluted 1 mL of the culture to an OD 600 value of 0.1 and prepared a series dilution. We then spread the final 3 dilutions of each sample onto plates and counted the CFUs. Because we started with an OD 600 value of 0.1, we know the OD values of the subsequent dilutions and can compare the resulting number of CFUs to our conversions from our cell measurement protocol.</p></div>
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