Background

Fluorescence data can not be directly compared, it is difficult to repeat the measurement. Different instruments, different settings and different measurement environments make this difficult. Through the Interlab study, we expect to have uniform fluorescence measurement steps and standards. These protocols solve this problem by establishing absolute units on the plate reader.

Design

OD 600 Reference point
Using LUDOX-S40 as a reference, the ratiometric conversion factor of Abs and OD600 can be obtained.
Protocol fluorescein fluorescence standard curve
By means of the gradient of the fluorescence standard curve, a method of obtaining a fluorescence correction of the cell can be obtained.
Cell measurement
At four time points, the fluorescence intensity of the eight circuits was measured.

Materials and Method

Plasmids used
Kit Plate 6 InterLab Part Locations
Positive Control (BBa_I20270): well 20B
Negative Control (BBa_R0040): well 20D
Test Device 1(BBa_J364000): well 20F
Test Device 2(BBa_J364001): well 20H
Test Device 3(BBa_J364002): well 20J
Test Device 4(BBa_J364003): well 20L
Test Device 5(BBa_J364004): well 20N
Test Device 6(BBa_J364005): well 20P
Strain
E.coli Competent DH5α （ TaKaRa Code : D9057）
Materials
1ml LUDOX (provided in kit) H20
Fluorescein (provided in kit) 10ml 1xPBS96 well plate, black with flat bottom
LB (Luria Bertani) media Chloramphenicol
50 ml Falcon tube Incubator at 37°C 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Pipettes
Machines
Tecan Spark™ 10M
Software
Microsoft Office EXCEL

Protocol

InterLab_2017_Plate_Reader_Protocol

Results

OD 600 Reference point

	LUDOX-HS40	H2O
Replicate 1	0.0497	0.0422
Replicate 2	0.0518	0.0495
Replicate 3	0.0546	0.0428
Replicate 4	0.0521	0.0569
Arith. Mean	0.05205	0.04785
Corrected Abs600	0.0042
Reference OD600	0.0425
OD600/Abs600	10.1190476

Table 1. OD600 Reference Point

Protocol fluorescein fluorescence standard curve

Figure 1.Fluorescein fluorescence standard curve

Cell measurement

	0h		2h		4h		6h
	Abs600	FI	Abs601	FI	Abs602	FI	Abs603	FI
Negative Control (Colony 1)	0.0301	7	0.1156	8	0.5106	14	0.7453	17
Negative Control (Colony 2)	0.0408	5	0.1352	8	0.5184	17	0.7326	16
Positive Control (Colony 1)	0.0308	28	0.0805	65	0.4715	476	0.7293	722
Positive Control (Colony 2)	0.0437	31	0.1498	136	0.4956	428	0.7049	677
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1)	0.0283	46	0.0883	152	0.4358	606	0.6846	948
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2)	0.0261	38	0.0969	119	0.3719	464	0.5960	738
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1)	0.0378	44	0.1120	164	0.5090	718	0.7145	1258
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2)	0.0223	16	0.0788	115	0.4045	506	0.5945	963
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1)	0.0323	1	0.0945	10	0.4482	22	0.5704	28
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2)	0.0156	4	0.0537	8	0.3399	18	0.5254	24
Test Device 4: J23101+I13504 (Colony 1)	0.0201	9	0.0585	76	0.3224	339	0.4975	472
Test Device 4: J23101+I13504 (Colony 2)	0.0257	16	0.0628	84	0.3267	340	0.4900	471
Test Device 5: J23106+I13504 (Colony 1)	0.0263	7	0.0804	46	0.4000	106	0.5413	120
Test Device 5: J23106+I13504 (Colony 2)	0.0269	6	0.0850	45	0.3789	94	0.5285	111
Test Device 6: J23117+I13504 (Colony 1)	0.0265	1	0.0959	8	0.3967	15	0.4837	2
Test Device 6: J23117+I13504 (Colony 2)	0.0243	-1	0.0883	5	0.4016	13	0.5212	10

Table 2. Raw data of Abs600 and Fluorescence measurement

	0h		2h		4h		6h
	average	SD	average	SD	average	SD	average	SD
Negative Control (Colony 1)	232	60	75	28	27	6	23	6
Negative Control (Colony 2)	114	11	59	25	34	4	22	3
Positive Control (Colony 1)	920	180	827	222	1013	75	994	87
Positive Control (Colony 2)	720	40	911	86	867	81	960	15
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1)	1636	324	1748	295	1394	80	1388	108
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2)	1462	64	1227	80	1248	64	1239	28
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1)	1190	212	1489	231	1413	69	1763	92
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2)	716	103	1469	163	1250	54	1621	83
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1)	27	61	112	50	49	7	49	11
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2)	282	108	148	45	52	3	45	11
Test Device 4: J23101+I13504 (Colony 1)	461	76	1319	170	1052	27	947	86
Test Device 4: J23101+I13504 (Colony 2)	617	136	1350	189	1042	73	962	51
Test Device 5: J23106+I13504 (Colony 1)	280	175	576	77	266	16	221	14
Test Device 5: J23106+I13504 (Colony 2)	215	93	530	41	249	20	211	12
Test Device 6: J23117+I13504 (Colony 1)	34	73	78	41	38	8	4	8
Test Device 6: J23117+I13504 (Colony 2)	-55	102	60	23	33	3	20	4

Table 3. Data of Fluorescence/Abs600

Figure 2.Average level of devices

Discussion

Device 2 and device 3 have strong fluorescence. Device 4 and device 5 have moderate fluorescence intensity. The device 3 and the device 6 are weakly fluorescent.