Team:KU Leuven/InterLabStudy/Results

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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.


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
Value Value Value Value Value Value Value Value Value Value Value

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

Figure 1
Restriction digest of the three devices with NcoI and XhoI.

Plasmid A Plasmid B

Figure 2
Left: plasmid A, Right: plasmid B. Click to enlarge

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