Team:KU Leuven/InterLabStudy/Results

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Interlab Results

The results of our Interlab Study are discussed in the section below.
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


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.

Terms used in the tables:
D1,D2,D3 -Devices J23117,J23106 and J23101 respectively
B1,B2,B3 -Biological replicates of the respective devices
T1,T2,T3 -Technical replicates of the respective devices

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 (D1, D2, D3) 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 (B1, B2, B3 of the respective devices).

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 (D1, D2, D3) 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 (T1, T2, T3 of the respective devices).

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
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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.

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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

Protocols

Detailed protocols and the worksheet can be found here.

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Protocols

Detailed protocols and the worksheet can be found here.
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Contact

Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be