1. Interlab Study

As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent E.coli DH5α. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD₆₀₀ and the fluorescence of the cells carrying the constructs. The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent DH5α cells.

1.1 Transformation of 8 test devices into competent DH5α cells

The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates. Therefore, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol over night for approximately 16-18 hours at 37°C and 220 rpm.

1.2 Measurement of LUDOX-HS40 OD600 Reference Point

The second segment is the measurement of the LUDOX-HS40 solution to obtain a ratiometric conversion factor which allows us to transform the absorbance data into a standard OD₆₀₀ measurement. This is necessary because plate readers in general do not have a 1 cm path length which means that measurements are volume dependent. By doing so, we obtained following data.

	LUDOX-HS40	H2O
Replicate 1	0,0543	0,0424
Replicate 2	0,0508	0,0413
Replicate 3	0,0515	0,0424
Replicate 4	0,051	0,042
Arith. Mean	0,0519	0,042
Corrected Abs600	0,009875
Reference OD600	0,0425
OD600/Abs600	4,303797

With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 μL of the provided LUDOX-HS40 to water.

1.3 Graphing a Fluorescein fluorescence standard curve

In the third segment we were instructed to measure a standard curve for fluorescence of Fluorescein which can be used to correct our cell based readings to an equivalent Fluorescein concentration. Following the instructions, we could obtain following results for the serial dilution of Fluorescein in phosphate buffered saline.

uM Fluorescein	50,00	25	12,5	6,25	3,125	1,5625	0,7813	0,3906	0,1953	0,0977	0,0488	0
Replicate 1	43791	28969	17384	9562	5001	2591	1355	676	344	173	87	3
Replicate 2	44517	29931	17243	9266	5010	2575	1325	668	324	170	84	2
Replicate 3	42992	28755	17684	10262	5451	2851	1439	733	369	184	91	2
Replicate 4	42990	28734	17280	9617	5261	2667	1381	696	337	178	90	5
Arith. Mean	43573	29097	17398	9676,8	5180,8	2671	1375	693,25	343,5	176,25	88	3
Arith. Std.Dev.	733,96	565,88	199,95	419,51	216,75	126,53	48,415	28,999	18,912	6,1305	3,1623	1,4142

uM Fluorescein/a.u.	50,00	25	12,5	6,25	3,125	1,5625	0,7813	0,3906	0,1953	0,0977	0,0488
Mean uM Fluorescein/a.u.	0,0011	0,0009	0,0007	0,0006	0,0006	0,0006	0,0006	0,0006	0,0006	0,0006	0,0006
Mean of med-high levels:	0,0007

These data give us an approximate linear regression line when plotted in a logarithmic scale and show an expected exponential course when plotted in a linear form.

1.4 Measurement of OD₆₀₀ and Fluorescence of Transformed Cells

In the last of the four segments the OD₆₀₀ and the fluorescence of the transformed cells were measured according to the protocol after 0, 2, 4 and 6 hours. Measurements of all these time points gave us the following data and calculations which were conducted with the values we obtained from the standard curves and the reference point.

Fluorescence Raw Readings:										Abs 600 Raw Readings:
Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)	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	228	262	294	371	227	275	249	230	221	Colony 1, Replicate 1	0,0473	0,0467	0,0478	0,0493	0,0483	0,0486	0,0474	0,0456	0,0435
Colony 1, Replicate 2	222	298	270	302	214	271	237	230	242	Colony 1, Replicate 2	0,0459	0,0466	0,0464	0,0468	0,0471	0,0474	0,0459	0,0442	0,043
Colony 1, Replicate 3	196	265	269	285	227	268	231	219	205	Colony 1, Replicate 3	0,05	0,0499	0,0502	0,0503	0,0518	0,0513	0,0502	0,0479	0,0473
Colony 1, Replicate 4	227	302	285	312	225	272	244	232	235	Colony 1, Replicate 4	0,061	0,0667	0,0691	0,0714	0,0725	0,0709	0,0596	0,0624	0,0412
Colony 2, Replicate 1	226	258	297	287	230	284	255	221	221	Colony 2, Replicate 1	0,0473	0,0471	0,0504	0,0484	0,0482	0,0482	0,0496	0,0482	0,0474
Colony 2, Replicate 2	217	277	301	311	221	283	244	244	231	Colony 2, Replicate 2	0,0469	0,0472	0,0492	0,0485	0,0473	0,047	0,0479	0,0474	0,0477
Colony 2, Replicate 3	213	261	287	285	222	276	236	225	234	Colony 2, Replicate 3	0,0512	0,0499	0,0563	0,0527	0,052	0,0523	0,0539	0,0533	0,0521
Colony 2, Replicate 4	223	267	294	303	224	293	249	238	243	Colony 2, Replicate 4	0,0604	0,0696	0,0686	0,0617	0,0719	0,0714	0,0608	0,0611	0,0646
Hour 2:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)	Hour 2:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	234	841	916	973	241	640	740	242	222	Colony 1, Replicate 1	0,0652	0,0568	0,0599	0,0613	0,068	0,0521	0,0742	0,075
Colony 1, Replicate 2	229	850	864	923	234	608	641	239	239	Colony 1, Replicate 2	0,06	0,0539	0,0569	0,057	0,0632	0,0496	0,0671	0,0713
Colony 1, Replicate 3	230	741	810	897	236	551	545	254	236	Colony 1, Replicate 3	0,0671	0,057	0,0626	0,0637	0,0671	0,0547	0,0698	0,0754
Colony 1, Replicate 4	227	758	942	893	240	564	613	241	240	Colony 1, Replicate 4	0,081	0,0695	0,082	0,0799	0,0833	0,0674	0,0836	0,085
Colony 2, Replicate 1	233	913	1016	1045	246	521	661	244	215	Colony 2, Replicate 1	0,0667	0,0602	0,062	0,065	0,0704	0,0499	0,0729	0,0774
Colony 2, Replicate 2	224	784	878	893	243	702	642	233	254	Colony 2, Replicate 2	0,0608	0,0558	0,0568	0,0592	0,0654	0,052	0,068	0,0721
Colony 2, Replicate 3	217	712	765	877	228	597	571	219	232	Colony 2, Replicate 3	0,0642	0,0614	0,0624	0,0669	0,0719	0,0584	0,0732	0,0676
Colony 2, Replicate 4	227	706	748	904	237	598	576	252	247	Colony 2, Replicate 4	0,0684	0,057	0,071	0,0803	0,0959	0,0653	0,0843	0,0848
Hour 4:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)	Hour 4:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6
Colony 1, Replicate 1	265	3725	4330	4534	327	1619	1493	293	224	Colony 1, Replicate 1	0,1765	0,1449	0,1431	0,1661	0,1677	0,0692	0,1766	0,193
Colony 1, Replicate 2	269	3980	3936	4087	334	1489	1551	275	229	Colony 1, Replicate 2	0,1804	0,1515	0,1365	0,1585	0,1861	0,07	0,19	0,167
Colony 1, Replicate 3	245	2579	3110	3080	300	1280	1257	276	238	Colony 1, Replicate 3	0,1321	0,1194	0,1199	0,132	0,1482	0,0666	0,1573	0,1578
Colony 1, Replicate 4	247	2934	3253	3214	313	1543	1358	304	234	Colony 1, Replicate 4	0,1354	0,1348	0,1296	0,1441	0,1762	0,0885	0,164	0,189
Colony 2, Replicate 1	269	3980	3936	4087	334	1489	1551	275	229	Colony 2, Replicate 1	0,3007	0,2746	0,2586	0,308	0,3246	0,339	0,3438	0,3446
Colony 2, Replicate 2	253	2825	3064	3547	321	1619	1294	308	237	Colony 2, Replicate 2	0,1467	0,1192	0,1135	0,1373	0,1643	0,0698	0,151	0,1849
Colony 2, Replicate 3	249	3397	3055	3125	309	1516	1238	276	239	Colony 2, Replicate 3	0,1374	0,1356	0,1199	0,1344	0,1453	0,0763	0,1532	0,1626
Colony 2, Replicate 4	261	2585	3302	3124	322	1589	1285	292	246	Colony 2, Replicate 4	0,1423	0,1255	0,138	0,1527	0,1686	0,0765	0,1522	0,1622
Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)	Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5
Colony 1, Replicate 1	289	7046	9753	10223	429	4924	5031	328	223	Colony 1, Replicate 1	0,2911	0,2572	0,2697	0,2843	0,311	0,1557	0,1581
Colony 1, Replicate 2	290	7161	9621	9685	429	4659	4916	322	233	Colony 1, Replicate 2	0,2735	0,2467	0,2558	0,2596	0,2984	0,1439	0,1471
Colony 1, Replicate 3	284	6900	8983	9620	420	4563	4830	335	236	Colony 1, Replicate 3	0,2694	0,2542	0,2557	0,2671	0,2856	0,1464	0,1531
Colony 1, Replicate 4	320	7664	10384	10759	441	5092	5472	341	239	Colony 1, Replicate 4	0,3314	0,2762	0,2711	0,2787	0,2999	0,1738	0,1763
Colony 2, Replicate 1	285	7246	9059	10726	445	2372	2368	332	229	Colony 2, Replicate 1	0,3007	0,2746	0,2586	0,308	0,3246	0,339	0,3438
Colony 2, Replicate 2	286	7418	10409	10079	442	2357	2321	329	232	Colony 2, Replicate 2	0,2853	0,2574	0,2783	0,2668	0,2989	0,3188	0,3134
Colony 2, Replicate 3	285	6939	9332	10013	439	2297	2299	336	232	Colony 2, Replicate 3	0,282	0,2546	0,2629	0,2691	0,3006	0,3064	0,3063
Colony 2, Replicate 4	294	7882	9938	10980	446	2523	2513	330	253	Colony 2, Replicate 4	0,2851	0,2633	0,2614	0,2668	0,2911	0,3239	0,336

2. Discussion

The protocol was easy to follow and the constructs were nicely expressed which allowed reliable and comparable measurements. In the process of obtaining transformed DH5α cells we did not encounter any problems with transformation as bacteria showed exceptional growth. The specific ratiometric conversion factor of our plate reader could be determined without further adjustments using the LUDOX-HS40 solution. When measuring the Fluorescein standard curve on a logarithmic scale the data points showed a linear distribution. The final step, the measurement of the fluorescence emission and optical density of the cells, resulted in the raw data shown in figure 1.5. To make this data visually accessible the mean values for OD and fluorescence were calculated and plotted against their respective time points which is seen in the following graphs.

Conclusively, these data suggest that the Interlab study was successful and that the provided protocol can be shared in the community for standardized fluorescence measurement with a plate reader.