Team:Gifu/InterLab

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INTERLAB

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[Introduction]
We joined InterLab Measurement Study in this year.The objective of this year’s iGEM Interlab study is to quantify expression of five different reporter constructs.

[Materials and Methods]
Materials
・Safire ART-NR:F129013
It is a plate reader to measure absorbance and fluorescence of samples.

Methods
1. Calculate the OD₆₀₀ Reference point
Measure absorbance 600 nm of LUDOX-S30 and H₂O in a 96 well plate in our plate reader. It is to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD₆₀₀ measurement.

2. Generate the FITC fluorescence standard curve
Prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in all standard modes, we can generate a standard curve of fluorescence for FITC concentration.

3. Measure cell
Day1 : Transform Escherichia coli these plasmids.
Plasmids
・Positive control
・Negative control
・Device1 : J23101 + I13504
・Device2 : J23106 + I13504
(We couldn’t transform E. coli with device3.)

Day2 : Pick 2 colonies from each of plate and glow the cells in LB medium + Chloramphenicol overnight. (37^○C and 220ppm)

Day3 : Cell growth, sampling, and assay
Measure OD₆₀₀ of these cells cultures and dilute it to a target OD₆₀₀ of 0.02 in 10 mL 0.5×TB medium + Chloramphenicol and incubate the cultures (37^○C and 220rpm).At 0,1,2,3,4,5,and 6 hours of incubation take 100 μL samples of the cultures. At the end of sampling point, measure Absorbance 600 nm and Fluorescence of these samples. (Use same instrument setting when measuring the FITC standard curve.)

[Result]
1. Calculate the OD₆₀₀ Reference point
The Abs₆₀₀ data of LUDOX and H₂O is Table1.
And correction factor to transform our absorbance data into a standard OD₆₀₀ measurement is calculated by the following formula.
Correction factor = Reference OD₆₀₀ / (LUDOX average – H₂O average)

   Table 1. Absorbance 600 nm of LUDOX-S30 and H₂O

	LUDOX	H2O
replicate 1	0.0485	0.0387
replicate 2	0.0482	0.0395
replicate 3	0.0579	0.0377
replicate 4	0.0589	0.0384
Average	0.0534	0.0386
Corrected Abs600	0.0148	---
Reference OD600	0.0148	---
Correction factor	0.0997	---

2. Generate the FITC fluorescence standard curve
The fluorescence data of FITC is table2. And FITC standard curve is figure1. We couldn’t measure the data of FITC concentration from 250 μM to 15.625 μM. Because we failed the settings.

   Table 2. fluorescence data of FITC in any dilution

Concentration (μM)	7.81	3.91	1.95	0.977	0.488	0.244	0
replicate 1	40211	20994	14525	7764	6858	4958	4502
replicate 2	46944	24730	15296	9009	7835	6234	5512
replicate 3	46384	26163	14271	9337	7858	4613	3915
replicate 4	45427	24076	12632	8586	5658	4273	2712
Average	44742	23991	14181	8674	7052	5020	4160
S.D.	3085	2180	1121	680	1040	857	1169

Fig.1


3. Measure cell　

  Table 3.

Sample	Abs600 reading	Volume of preloading culture	Volume of preloading media
Positive control	0.0799	9.26	0.741
Negative control	0.0690	18.7	-8.69
Device1	0.0814	8.66	1.34
Device2	0.0805	9.01	0.991
Media+chl	0.0583	---	---

   Table 4. blank substraction and correction (bacteria concentration)

	Positive control 1	Positive control 2	Negative control 1	Negative control 2	Device 1-1	Device 1-2	Device 2-1	Device 2-2
0 h	0.02040	0.01861	0.09539	0.01392	0.02190	0.021	0.009639	0.01113
1 h	0.04103	0.03934	0.02568	0.03087	0.02889	0.02758	0.02758	0.02897
2 h	0.1035	0.1014	0.09595	0.1029	0.05598	0.5349	0.09834	0.09804
3 h	0.1832	0.1841	0.1625	0.1789	0.1073	0.1063	0.1608	0.1702
4 h	0.3287	0.3313	0.3176	0.3394	0.1875	0.1810	0.3076	0.3090
5 h	0.4961	0.5037	0.4722	0.5253	0.3389	0.3310	0.4858	0.4858
6 h	0.7103	0.7191	0.6908	0.7418	0.5524	0.5456	0.7116	0.7067

Fig.2

  Table 5. blank substraction (fluorescence)

null	Positive control 1	Positive control 2	Negative control 1	Negative control 2	Device 1-1	Device 1-2	Device 2-1	Device 2-2
0 h	4550	5376	1488	941	18215	18519	3956	4360
1 h	6166	6942	414	294	18064	18362	3688	4091
2 h	18428	19780	-426	-255	26946	27597	6800	7414
3 h	25619	27051	-495	-340	33766	35092	10233	10839
4 h	32258	34499	-652	-480	50905	51913	14704	15534
5 h	47617	50169	-802	-494	-8413	-8413	25032	25931
6 h	-8413	-8413	-693	-439	-8413	-8413	35887	35386

Fig.3

  Table 6. Value of Flu/Abs₆₀₀

null	Positive control 1	Positive control 2	Negative control 1	Negative control 2	Device 1-1	Device 1-2	Device 2-1	Device 2-2
0 h	223025	288914	156012	67595	831851	881857	410449	391624
1 h	150277	176476	16127	9532	625632	665830	133738	141207
2 h	178015	195018	-4438	-2476	481340	515931	69151	75625
3 h	139881	146900	-3044	-1900	314667	330090	63629	63687
4 h	98152	104118	-2052	-1414	271443	286880	47799	50269
5 h	95985	99608	-1698	-940	-24822	-25413	51525	53376
6 h	-11845	-11699	-1003	-592	-15230	-15419	50435	50074

  Table 7. Average and S.D.

Average	Positive control	Negative control	Device 1	Device 2	S.D.	Positive control	Negative control	Device 1	Device 2
0 h	255970	1118034	856854	401036	0 h	46591	62520	35360	13311
1 h	163376	12829	645731	137472	1 h	18525	4664	28424	5281
2 h	186517	-3457	498636	72388	2 h	12023	1388	24460	4578
3 h	143390	-2472	322379	63658	3 h	4963	809	10906	41
4 h	101135	-1733	279161	49034	4 h	4219	452	10916	1747
5 h	97797	-1319	-25117	52450	5 h	2562	536	417	1308
6 h	-11772	-797	-15324	50254	6 h	103	291	134	255

Fig4.