Team:KU Leuven/InterLabStudy/Results

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Research

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Future

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Interlab Results

The results of our Interlab study are discussed in this section
The minipreped samples of the given devices were validated by restriction mapping using the enzymes NcoI and XhoI. These restriction enzymes left us with bands around 267, 364, 625 and 1724 basepairs. This was validated by a gel electrophoresis (figure 1). The numbers 101, 106 and 117 stand for the devices containing the promoters J23101, J23106 and J23117 respectively.

Figure 1

Restriction digest of the three devices with NcoI and XhoI. Click to enlarge

The restriction digest (Fig 1) shows our devices containing GFP I13504 and the promoters J23101, J23106 and J23117.The expected bands were at 267, 364, 625, 1724 bp respectively.

We made a Fluorescein standard graph and extrapolated the concentration of the GFP from the samples using the fluorescence and the absorbance values that were recorded. We went ahead with those values to calculate the mean and the standard deviation for our biological and technical replicates. We processed all the data in Microsoft excel.

Table 1: The raw fluorescence data ( excitation at 483 nm and emission at 525 nm) for LB medium containing chloramphenicol, LB medium with chloramphenicol and cells containing the biobrick J3101 and the three devices (D1, D2, D3). Biological replicates originating from three different devices are presented in the rows and the technical replicates for the devices are presented in the columns.

LB+Cam	LB+Cam+Cells	D1			D2			D3
512	601	737	685	751	9425	9322	9737	22786	25895	25048
546	594	754	641	641	9669	9545	9876	23159	26460	24785
549	587	722	660	697	9407	9321	9677	22719	25931	24935

Table 2: Absorbance values measured by the Tecan Safire2 plate reader at 600 nm (O.D. within 5% of 0.5 in a cuvette with a path length of 1 cm). The absorbance depends on the path length which is different in our plate reader attributing to the values lower than 0.5.

LB+Cam	LB+Cam+Cells	D1			D2			D3
0.0434	0.2052	0.2174	0.1942	0.2201	0.2088	0.2082	0.2154	0.2139	0.2210	0.2218
0.0405	0.2000	0.2182	0.1974	0.1980	0.2108	0.2111	0.2123	0.2143	0.2179	0.2056
0.0379	0.2064	0.2141	0.1961	0.2036	0.2137	0.2117	0.2164	0.2202	0.2261	0.2083

Table 3: Fluorescence values divided by the absorbance values for the biological and technical replicates of our three devices and for our negative control (LB medium with kanamycin and cells containing the promoter J3101), the arithmetic average and the standard deviation of the biological replicates.

	D1			D2			D3
	T1	T2	T3	T1	T2	T3	T1	T2	T3
B1	3390	3456	3372	45139	45868	44020	1065826	108068	103174
B2	3527	3247	3366	44774	45216	44029	117172	121432	114688
B3	3399	3237	3426	45204	46519	44718	112931	120550	119707
Brep Avg	3439	3313	3388	45039	45868	44256	112210	116683	112523
BRep SD	270	101	162	189	532	327	4376	6102	6921

Table 4: Fluorescence values divided by the absorbance values for the biological and technical replicates of our three devices and for our negative control (LB medium with kanamycin and cells containing the promoter J3101), the arithmetic average and the standard deviation of the technical replicates.

	D1			D2			D3			LB+Cam+Cells
	B1	B2	B3	B1	B2	B3	B1	B2	B3
T1	3390	3527	3399	45139	44774	45204	106526	117172	112931	2929
T2	3456	3247	3237	45868	45216	46519	108068	121432	120550	2970
T3	3372	3366	3426	44020	44029	44718	103174	114688	119707	2844
TRep Avg	3406	3380	3354	45009	44673	45480	105923	117764	117729	2914
TRep SD	36	115	140	760	490	761	2043	2785	3410	52

Table 5: Fluorescein standard curve. F1, F2 and F3 represent three technical replicates of every sample. From these technical replicates, we calculated the average F and the standardised average F’. The standardised average F’ is equal to the difference of the respective sample and the average F with a concentration of 0 ng/ ml fluorescein.

Concentration (ng/mL)	F1	F2	F3	average F	average F'	SD
0	9	13	12	11	0	1.699
10	1140	1168	1151	1153	1142	11.518
25	2543	2600	2631	2591	2580	36.445
50	4822	5097	5154	5024	5013	144.951
125	12879	12902	12931	12904	12893	21.276
250	25494	25761	25765	25673	25662	126.818
375	37474	37505	37899	37626	37615	193.454
500	46698	49051	48798	48182	48171	1054.652

Figure 2

Fluorescein standard curve with varying concentrations of fluorescein: 0, 10, 25, 50, 125, 250, 375 and 500 ng/ mL. The trendline and R2 is based on the standardised values. The error bars show the variation among the technical replicates.

Table 6 : Extrapolation of GFP concentration using fluorescence measurements and fluorescein standard.

	D1			D2			D3
Brep Avg	3439	3313	3388	45039	45868	44256	112210	116683	112523
(Brep Avg) - (LB+cam+cells avg)	525	399	474	42125	42954	41342	109296	113769	109609
Concentration (ng/mL)	1.929	0.639	1.407	427.88	436.36	419.88	1115.64	1161.44	1118.85
TRep Avg	3406	3380	3354	45009	44673	45480	105923	117764	117729
(Trep avg) - (LB+cam+cellsavg)	492	466	440	42095	41759	42566	103009	114850	114815
Concentration (ng/mL)	1.591	1.325	1.059	427.56	424.12	432.39	1051.27	1172.51	1172.15

The figure 3 represents a summary of our results. The blue bars represent the average of the biological replicates and the orange bars represent the average of the technical replicates. The error bars indicated represent the standard deviation of the corresponding average. J23117 (device 1) clearly shows that the strength of the promotor is weak, while J23106 (device 2) has a promotor with medium strength and J23101 (device 3) has the strongest promotor.

Figure 3

Graph representing the summary of our results per device. The biological and technical replicates of the three devices are shown. The error bars represent the standard deviations.Click to enlarge