Team:USP-Brazil/InterLab

Wiki - iGEM Brazil

Interlab

The iGEM Interlab Project is the biggest interlaboratory studies in the world, and it is now on its fifth edition. This project aims to improve a protocol to measure fluorescence and minimize the variation between assays. Improved reproducibility will allow research groups to share data more safely. And to achieve this goal, our iGEM Team makes an effort to refine and test multiple protocols since 2016, trying to standardize every step, in a way that the results will be accurate regardless of the outside variables. This year, the focus was on improving the strategy to normalize the fluorescence by the population size. For that, teams around the globe compared the results of cell count utilizing two methods, the easier, using optical density (OD), and a more direct one, based on colony formation units (CFU).

We have worked with the devices made available by iGEM, and strictly followed the available protocols. The measurements were taken with a plate reader, and the results were sent to iGEM headquarters. The preliminary analysis showed that the results behaved as expected. Being that, we have successfully contributed to this year Interlab Study! Our team was happy to participate in this daring project, and our experience is further detailed below.

Calibration Procedures

Three calibration protocols were followed before the cell measurements, one for fluorescence setups and the others for optical density. The first was conducted using increasing concentrations of fluorescein, creating a standard fluorescence curve. The calibration for absorbance followed the same thought, but this time doing a standard curve using silica microspheres that resemble Escherichia coli cells. Together with a LUDOX analises, this can be successfully used to standardize OD measurements. Thus, this curves will help in the analysis of our data, by correcting the results and enable comparison.

Cell Measurements

The first step was to transform E.coli DH5α with the test devices provided by iGEM, which are listed below:

Device	Part Number	Plate	Location
Negative control	BBa_R0040	Kit Plate 7	Well 2D
Positive control	BBa_I20270	Kit Plate 7	Well 2B
Test Device 1	BBa_J364000	Kit Plate 7	Well 2F
Test Device 2	BBa_J364001	Kit Plate 7	Well 2H
Test Device 3	BBa_J364002	Kit Plate 7	Well 2J
Test Device 4	BBa_J364007	Kit Plate 7	Well 2L
Test Device 5	BBa_J364008	Kit Plate 7	Well 2N
Test Device 6	BBa_J364009	Kit Plate 7	Well 2P

Two colonies with each of the devices were selected and grown overnight in liquid LB medium. They were used to make the inoculum on time 0 of the assay. Then, the cultures were incubated at 37°C and 220 rpm for 6 hours. An aliquot was taken from each colonie at timepoints 0h and 6h, which were used to do the OD and fluorescence measurements. The raw data we obtained are represented in the table below:

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	4134783	8620888	4276694	4264055	4276338	26381838	29354062	8782100	4235415
Colony 1, Replicate 2	4454708	8441827	4402544	4284872	4123260	25604070	28618510	9595188	4331713
Colony 1, Replicate 3	4116980	8632287	3948719	4006743	3974325	26771162	25442728	9131540	4035518
Colony 1, Replicate 4	4129309	8186708	3887090	3982249	4196195	25012198	28590348	9608511	4214761
Colony 2, Replicate 1	3816828	8303402	3870684	3824678	4087630	33446040	16690344	7320758	4134598
Colony 2, Replicate 2	4173630	8565011	4110887	3709872	4297948	35265600	25184870	7866042	4045934
Colony 2, Replicate 3	4065128	8202472	4000656	4101600	4456035	34174844	26750768	7892292	3926784
Colony 2, Replicate 4	3848680	8052030	4040516	4088390	4468382	39613912	27035656	8328709	4026642

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	5250460	207554160	5110779	5434859	10324422	151222032	88983128	140674640	3327864
Colony 1, Replicate 2	5623197	206436368	5797002	5441559	10779428	142598096	86583136	146841856	3207918
Colony 1, Replicate 3	5443169	218250752	5831971	5393474	10452652	160754688	85738744	151173840	3271237
Colony 1, Replicate 4	5810695	217608752	5950365	5324008	10633720	140962736	77727336	120463808	3368912
Colony 2, Replicate 1	5299750	178081840	5668620	5076567	7952136	210299040	51507120	82873880	3438758
Colony 2, Replicate 2	5178408	176944672	4995087	5028444	7840034	189582448	52489372	88725576	3330172
Colony 2, Replicate 3	5289134	167250016	5143092	5016710	8425689	197933584	59037472	87584376	3404846
Colony 2, Replicate 4	5064710	178135616	5420042	5299285	8464418	204021472	56090556	91463760	3643057

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	0.326	0.1921	0.2807	0.2736	0.223	0.264	0.2436	0.2501	0.2426
Colony 1, Replicate 2	0.2808	0.2474	0.2683	0.2849	0.2626	0.246	0.2539	0.2829	0.229
Colony 1, Replicate 3	0.29	0.2851	0.2581	0.3023	0.2641	0.256	0.305	0.2815	0.2585
Colony 1, Replicate 4	0.3081	0.277	0.3172	0.3263	0.314	0.2826	0.2658	0.2972	0.2623
Colony 2, Replicate 1	0.3295	0.2748	0.2841	0.3171	0.298	0.3073	0.3154	0.2976	0.2839
Colony 2, Replicate 2	0.2998	0.2936	0.3135	0.3247	0.2948	0.315	0.3027	0.328	0.2614
Colony 2, Replicate 3	0.3226	0.2782	0.292	0.307	0.3113	0.3001	0.2926	0.3097	0.3296
Colony 2, Replicate 4	0.315	0.2981	0.3032	0.2873	0.3113	0.2905	0.3265	0.3579	0.2944

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	0.9903	0.8532	0.9722	1.0118	0.8718	0.3918	0.3633	0.8469	0.2673
Colony 1, Replicate 2	1.0088	0.8578	1.0146	1.0322	0.8874	0.436	0.3899	0.8822	0.2622
Colony 1, Replicate 3	1.0063	0.8659	1.0253	1.0268	0.9018	0.4389	0.416	0.8878	0.3025
Colony 1, Replicate 4	1.0181	0.8748	1.0331	1.0315	0.8938	0.4308	0.3903	0.826	0.2517
Colony 2, Replicate 1	0.9871	0.8087	0.911	0.961	0.8131	0.4632	0.3922	0.7132	0.307
Colony 2, Replicate 2	0.955	0.7853	0.8715	0.9531	0.7955	0.4529	0.3462	0.7247	0.2367
Colony 2, Replicate 3	0.9649	0.7887	0.8895	0.9399	0.808	0.4615	0.3626	0.7182	0.2422
Colony 2, Replicate 4	0.9694	0.7809	0.8847	0.9726	0.784	0.4152	0.3174	0.7136	0.2294

Normalized Data

Flourescence per OD:

Flourescence per Particle:

Colony Formation Units

This step was necessary to calibrate our OD measurements with CFU counts, and with that, obtain a more reliable number of cells. First, we measured the absorbance of the overnight cultures from the two controls. Them, we diluted each one to a 0.1 OD in triplicate, and measure again. That value will be compared to the CFU counts from the same cultures. To obtain a countable number of colonies each inoculum was dillute 8 x 10⁴, 8 x 10⁵ and 8 x 10⁶ times before being placed on an agar plate. The plates were incubated overnight and the number of colonies were computed and sent with our relatory.