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Revision as of 01:31, 18 September 2015
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
Contact
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Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be
