The interlab is aimed to create reliable and repeatable measurement data and standardize the measurement of fluorescence in labs around the world by testing replicability of data, thus achieving to support the engineering of biological experiment. This academic community prevents the disadvantages of difference in experiment by providing a detailed protocol and data analysis method. Interlab has become one of the largest interlaboratory study worldwide in synthetic biology, hitherto. The data collected from the previous studies is summarized in a presentation iGEM 2016 Interlab Results Presentation. This time it is done by following the same protocols designed for a plate reader and a flow cytometer that will produce data in same units everywhere so it can be compared and analysed. Our team decided to test both protocols to measure the fluorescence.

Plasmids used

Positive Control Device: I20270 in pSB1C3 (Located in Kit Plate 6, well 20B)

Negative Control Device: R0040 in pSB1C3 (Located in Kit Plate 6, well 20D)

• Test Device 1: J23101+I13504 in pSB1C3 (Located in Kit Plate 6, well 20F)

• Test Device 2: J23106+I13504 in pSB1C3 (Located in Kit Plate 6, well 20H)

• Test Device 3: J23117+I13504 in pSB1C3 (Located in Kit Plate 6, well 20J)

• Test Device 4: J23101.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20L)

• Test Device 5: J23106.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20N)

• Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)

Strain used

• Escherichia coli​ strain DH5α

Materials

• 1ml LUDOX

• H​2​0

• 96 well plate

• fluorescein

• 10ml 1xPBS

• LB (Luria Bertani) media

• Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)

• 50 ml Falcon tube

• Incubator at 37°C

• 1.5 ml eppendorf tubes

• Ice bucket with ice

• Pipettes

Machines

• Thermo Multiskan FC

• BERTHOLD TECHNOLOGIES LB940Mith

Calibration Protocols Cell measurement protocol

• Positive Control ：BBa_I20270

• Negative Control ：BBa_R0040

• Test Device 1 ：BBa_J364000

• Test Device 2 ：BBa_J364001

• Test Device 3 ：BBa_J364002

• Test Device 4 ：BBa_J364003

• Test Device 5 ：BBa_J364004

• Test Device 6 ：BBa_J364005

> BBa_I20270 Part-only sequence (919 bp)

ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

> BBa_R0040 Part-only sequence (54 bp)

tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac

> BBa_J364000 Part-only sequence (918 bp)

tttacagctagctcagtcctaggtattatgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

> BBa_J364001 Part-only sequence (918 bp)

tttacggctagctcagtcctaggtatagtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

> BBa_J364002 Part-only sequence (918 bp)

ttgacagctagctcagtcctagggattgtgctagctactagagaaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

> BBa_J364003 Part-only sequence (990 bp)

tttacagctagctcagtcctaggtattatgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

> BBa_J364004 Part-only sequence (990 bp)

tttacggctagctcagtcctaggtatagtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

> BBa_J364005 Part-only sequence (990 bp)

ttgacagctagctcagtcctagggattgtgctagctactagaggggcccaagttcacttaaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggttttcttactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

OD600 Reference Point

Table 1. OD600 Reference Point.

	LUDOX-HS40	H2O
Replicate 1	0.0497	0.0483
Replicate 2	0.0493	0.0463
Replicate 3	0.0518	0.0492
Replicate 4	0.0516	0.047
Arith. Mean	0.0506	0.0477
Corrected Abs600	0.0026667
Reference OD600	0.0425
OD600/Abs600	15.9375

Fluorescein Standard Curve

Table 2. Data of Fluorescein Standard Curve.

uM Fluorescein	50.00	25	12.5	6.25	3.125	1.5625	0.78125	0.390625	0.195313	0.097656	0.048828	0
Replicate 1	1937386	1040680	592161	322379	163819	86765	42958	22216	11739	5925	3079	444
Replicate 2	1859453	1115787	617759	316051	158096	82081	42161	21509	11052	5854	3048	444
Replicate 3	1874906	1099274	611612	327265	156925	77801	40314	19642	10255	5430	2655	373
Replicate 4	1893953	1082973	639359	340346	172348	86573	44775	21540	11446	6076	3351	414
Arith. Mean	1891425	1084679	615222.8	326510.3	162797	83305	42552	21226.75	11123	5821.25	3033.25	418.75
Arith. Std.Dev.	33733.71	32246.67	19441.13	10303.09	7043.812	4260.032	1850.053	1105.718	643.4931	276.7735	286.5593	33.61919

Normalization

Table 3. Normalization.

sample	Abs600 Reading	Volume of Preloading Culture	Volume of Preloading Media
positive control	0.090625	6.441223833	3.558776167
negative control	0.0985875	5.126561999	4.873438001
device 1	0.09785	5.225342913	4.774657087
device 2	0.10105	4.822182037	5.177817963
device 3	0.103175	4.587155963	5.412844037
device 4	0.0987375	5.106926269	4.893073731
device 5	0.1183625	3.402083776	6.597916224
device 6	0.0985	5.138086063	4.861913937
media+chl	0.059575

Cell Measurement

Table 4. Raw data of Abs600 measurement.

Abs600 Raw Readings:	Neg. Control		Pos. Control		Device 1		Device 2		Device 3		Device 4		Device 5		Device 6		LB + Chlor (blank)
	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2
Hour 0:	0.09155	0.0897	0.090325	0.10685	0.104875	0.090825	0.09655	0.10555	0.1096	0.09675	0.107025	0.09045	0.13085	0.105875	0.1069	0.0901	0.0664	0.059575
Hour 2:	0.1793	0.1855	0.10325	0.16325	0.10525	0.11365	0.17545	0.15035	0.202875	0.184825	0.1497	0.1019	0.19725	0.177475	0.1877	0.160825	0.0683	0.05675
Hour 4:	0.2421	0.246575	0.134175	0.249425	0.117225	0.208075	0.23985	0.161875	0.4247	0.25865	0.230525	0.124025	0.25595	0.240925	0.244225	0.24665	0.06815	0.06885
Hour 6:	0.3424	0.288625	0.208925	0.35995	0.133325	0.360225	0.37615	0.20765	0.58355	0.3692	0.303125	0.16655	0.36025	0.284425	0.291575	0.3055	0.0779	0.064775

Table 5. Blank subtraction and correction of Abs600 measurement.

OD600-Background	Neg. Control		Pos. Control		Device 1		Device 2		Device 3		Device 4		Device 5		Device 6
	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2
Hour 0:	0.02515	0.0233	0.023925	0.04045	0.038475	0.024425	0.03015	0.03915	0.0432	0.03035	0.040625	0.02405	0.06445	0.039475	0.0405	0.0237
Hour 2:	0.1129	0.1191	0.03685	0.09685	0.03885	0.04725	0.10905	0.08395	0.136475	0.118425	0.0833	0.0355	0.13085	0.111075	0.1213	0.094425
Hour 4:	0.1757	0.180175	0.067775	0.183025	0.050825	0.141675	0.17345	0.095475	0.3583	0.19225	0.164125	0.057625	0.18955	0.174525	0.177825	0.18025
Hour 6:	0.276	0.222225	0.142525	0.29355	0.066925	0.293825	0.30975	0.14125	0.51715	0.3028	0.236725	0.10015	0.29385	0.218025	0.225175	0.2391

Figure 3. Blank subtraction and correction of Abs600 measurement.

Table 6. Raw data of fluorescence measurement.

Raw fluorescence	Neg. Control		Pos. Control		Device 1		Device 2		Device 3		Device 4		Device 5		Device 6		LB + Chlor (blank)
	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2
Hour 0:	11978.75	12054.25	13919.25	14055.25	12311.5	13868.5	17252.5	14736.75	12430.25	12286.5	14287.5	13227.5	12238.25	12178.25	11854.5	12076.75	11317.25	11138.25
Hour 2:	12564	12755.75	16652	22236.5	15541.5	15458.5	24010.25	17610.75	13101.5	12591.75	20517.75	14232	14340.5	14186.5	12589.5	12354.75	10681.75	10787.5
Hour 4:	13356.25	13479.75	21582.75	31126.25	13257.75	20684.25	28890.5	18862.25	14835	13921.5	28713.5	15357.25	15024.5	15387.5	13588.5	13468.25	11143.25	11640.5
Hour 6:	14065.75	13709.75	27002.75	39536.5	14482	27169.5	38477	22481	17517.5	14774.75	32347.5	17138.75	16798.5	15973.25	14512	14396	11572.25	11271.75

Table 7. Blank substraction and correction of fluorescence measurement.

fluorescence-Background	Neg. Control		Pos. Control		Device 1		Device 2		Device 3		Device 4		Device 5		Device 6
	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2
Hour 0:	661.5	737	2602	2738	994.25	2551.25	5935.25	3419.5	1113	969.25	2970.25	1910.25	921	861	537.25	759.5
Hour 2:	1246.75	1438.5	5334.75	10919.25	4224.25	4141.25	12693	6293.5	1784.25	1274.5	9200.5	2914.75	3023.25	2869.25	1272.25	1037.5
Hour 4:	2039	2162.5	10265.5	19809	1940.5	9367	17573.25	7545	3517.75	2604.25	17396.25	4040	3707.25	4070.25	2271.25	2151
Hour 6:	2748.5	2392.5	15685.5	28219.25	3164.75	15852.25	27159.75	11163.75	6200.25	3457.5	21030.25	5821.5	5481.25	4656	3194.75	3078.75

Figure 4. Blank subtraction and correction of fluorescence measurement.

Table 8.Raw data of Fl/Abs600.

FI/OD600	Neg. Control		Pos. Control		Device 1		Device 2		Device 3		Device 4		Device 5		Device 6
	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2	Colony 1	Colony 2
Hour 0:	0.0363656	0.0436894	0.1610903	0.1046872	0.0371617	0.2434395	0.3112388	0.1224077	0.0351709	0.0459574	0.1034099	0.1136499	0.0257567	0.0291739	0.0207388	0.0473831
Hour 2:	0.015015	0.0165508	0.1993033	0.1555274	0.1629687	0.1344471	0.1612764	0.1036844	0.0178552	0.0149248	0.1559002	0.1161702	0.0335034	0.0354444	0.0141051	0.0152842
Hour 4:	0.0159176	0.016459	0.2081384	0.1484984	0.0537376	0.0917684	0.1392104	0.1085667	0.0134581	0.018553	0.1457091	0.0972347	0.0276245	0.0320685	0.017551	0.0165957
Hour 6:	0.0136599	0.014732	0.1650095	0.1317819	0.0655606	0.0739985	0.1257605	0.108465	0.0163216	0.016774	0.1236002	0.0795852	0.0257259	0.0296143	0.0193078	0.017636

Table 9. Raw data of average

Raw data of average	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Hour 0:	0.04002752	0.132888796	0.140300608	0.216823275	0.040564166	0.108529872	0.027465311	0.034060932
Hour 2:	0.015782922	0.177415362	0.148707896	0.132480415	0.016390008	0.108529872	0.034473902	0.014694671
Hour 4:	0.016188308	0.178318419	0.072753021	0.123888542	0.016005559	0.108529872	0.029846506	0.01707336
Hour 6:	0.014195916	0.148395679	0.069779519	0.117112747	0.016547803	0.108529872	0.027670075	0.018471905

Table 10. Raw data of SD

Raw data of SD	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Hour 0:	0.015079549	0.05186329	0.107849912	0.07821561	0.008373059	0.02479548	0.008997379	0.021020162
Hour 2:	0.002464455	0.011865598	0.047550627	0.014623325	0.00337995	0.029521958	0.005492679	0.003828995
Hour 4:	0.001357521	0.009555002	0.012588667	0.006161179	0.00202254	0.011469346	0.004159737	0.002278645
Hour 6:	0.002069122	0.029215462	0.005424471	0.012271926	0.003021193	0.00655286	0.003027256	0.002185333

Figure 5. Average level of devices.