Team:Bielefeld-CeBiTec/InterLab

Interlab Study

Reproducibility of results in different laboratories is crucial to facilitate science. We participated in the interlab study to support the iGEM community with more data for interlab comparison of measurement results.

Protocol

We applied the protocol for plate reader that was provided by the iGEM HQ.
This year, in addition to the positive and negative control, six other plasmids containing the GFP gene, different ribosome binding sites and varying Anderson promoters were transformed into Escherichia coli.
The following devices were tested:

positive control (BBa_I20270): GFP under control of a weak Anderson promoter
negative control (BBa_R0040): TetR repressible promoter
TD 1 (BBa_J364000): J23101 + I13504
TD 2 (BBa_J364001): J23106 + I13504
TD 3 (BBa_J364002): J23117 + I13504
TD 4 (BBa_J364003): J23101.BCD2.E004.B0015
TD 5 (BBa_J364004): J23106.BCD2.E004.B0015
TD 6 (BBa_J364005): J23117.BCD2.E004.B0015

In the beginning, there were complications to transform the plasmids directly from the distribution plates. Therefore, we transformed them to E. coli XL-1 Blue using 1 µL plasmid each from kit plate 6 and 7. The colonies were picked and incubated overnight in 5 mL LB and 0.25 µL mL^-1 chloramphenicol. After the plasmid isolation, all parts were sequenced. Plasmids with correct sequences were then retransformed in E. coli DH5-α.

For measurement two cultures of each plate were picked and grew over night in 5 mL LB media with 0.25 µL mL^-1 chloramphenicol.

The bacteria cultures were diluted to OD₆₀₀ of 0.02 in a total volume of 24 mL LB with chloramphenicol in 250 mL shake flasks and incubated without light for 6 hours at 37 °C and 220 rpm. 500 µL samples were taken after 0, 2, 4 and 6 hours of incubation and stored on ice until the measurement. The temperature during the measurement was at least 21.6 °C .

Material

Incubator: We used the New Brunswick^TN Innova^®43/43R at 37 °C and 220 rpm. We covered the glass front with paper to make sure the samples grew in darkness.

Cuvette measurement: For the first OD₆₀₀ measurement of the overnight cultures we used an Eppendorf 6131 BioPhotometer.

Plate reader: Our plate reader model was the Tecan Infinite M200 (Roche). The excitation wavelength was set to 488 nm and emission wavelength was detected at 510 nm [1].

96 well plate: We used the “Nunc^TM MicroWell^TM 96-Well PolymerBase Black” by Thermo Fisher Scientific for our measurement with the plate reader.

E. coli strains

E. coli DH5-α

E. coli XL-1 Blue

Results

After 6 hours of cultivation and storage on ice, the optical density of all cultures with the different plasmids was measured.

Figure 1: Development of the OD₆₀₀ of the tested devices over the cultivation time of six hours. The measured samples were test devices (td) 1-6, the positive control (pos) and negative control (neg).

The OD₆₀₀ values increased during the six hours of cultivation, except for test device 1, where both colonies did not persist the dilution (Figure 1).

Figure 2: Fluorescence of the tested devices signal over the cultivation time of six hours. The measured samples were test devices (td) 1-6, the positive control (pos) and negative control (neg).

The mean fluorescence does not correspond to the expected results. Proportional to the increasing OD₆₀₀ values, we expected all fluorescence intensities to rise. But none of the cultures showed an exponential increase. Although test device 1 did not survive the cultivation, it had the strongest fluorescence during the first four hours.

Figure 3: Comparison of the mean fluorescence of the tested devices with N = 18. The measured samples were test devices (td) 1-6, the positive control (pos) and negative control (neg).

As can be seen in figure 3 the mean fluorescence of the positive control is the strongest during the whole cultivation. In contrast to our expectation the negative control shows a stronger mean fluorescence than all test devices except test device 1.

In the final analysis, all measured values in general did not fulfil the expectation. The maximal OD₆₀₀ values did not reach the 0.7 and although all cells (except test device 1) did grow steadily, the fluorescence curve is not uniform.

Due to theses unexpected results, we repeated the whole process to validate our findings. Moreover, all plasmids were checked by Sanger sequences to exclude the influence of sequence variants. No sequence errors were detected in the plasmids.

Since we are measuring different fluorescence signals on a routine basis, we are certain that the hardware in our lab is fine.