Team:BIT/interlab.html

Our team are competing in the Measurement Track of iGEM this year, and involved in Interlab study according to the requirements.

The Interlab study contributes to enrich the data of three specific GFP expression devices from iGEM teams all over the world.

With the DH5a as an expression host, we transfer the three devices into E.coli cells separately. And we measure the fluorescence data and optical density data with spectral scanning machine every 1 hour during a 8 hours cultivation.

We use the Thermo Scientific Varioskan Flash spectral scanning to measure. We could show that the fluorescence we measured is rather a function of each cell than the whole culture, since all cultures had comparable optical densities.

Experimental Design

For the Interlab Part, we tested GFP-containing BioBricks for fluorescence and OD. We use these parts of BioBricks, I20260, J23101+E0240 and J23115+E0240. The I20260 is settled in a pSB3K3 with an insert set, which consists of the combination of the promoter J23101, the RBS B0032, the GFP coding sequence E0040 and the terminator B0015. The second part has the same insert as I20260, but uses pSB1C3 as a carrier. There is only one difference between the latter two that J23115+E0240 using another promoter called J23115. As a negative control, we introduce a dilution set of Fluorescein sodium in gradient: 10-7 ,8*10-8, 4*10-8, 2*10-8, 1*10-8mol/L, and set of dilution with a no-fluorescent-expression culture.

We incubate the bacteria in a constant temperature 37℃ for 12 hours, then we measure optical density and fluorescence per hour using the Thermo Scientific Varioskan Flash spectral scanning.

We use endonuclease (EcoRI and PstI, XbaI and PstI) and T4 DNA ligase to construct them. Finally, the ligation product was introduced into the pSB1C3. All constructs used were transformed into DH5α cells.

Experimental Methods

The cultivation of our bacteria was performed in tubes filled with 5 mL LB medium. The cultures were kept at 37 °C and 150 rpm in shaker. Antibiotics were added to each media (kanamycin 58 µg/mL for I20260, chloramphenicol 170 µg/mL for B0015, J23101.E0240 and J23115.E0240).

We incubate the bacteria in a constant temperature for 12 hours , then we put 50μL cultivated mass in tubes per hour, each group keeps three parallel samples. After obtaining eight or more gradients, we transfer the culture liquid 150μL per tube into a 96-wells plank to do the measurement.

Measurement of fluorescence and OD were performed using the Thermo Scientific Varioskan Flash with the 'Thermo Scientific SkanIt' software.

Expected Results

Fluorescence was expected to increase in series containing I20260, J23101.E0240 and J23115.E0240 along with time, because them all include the GFP coding sequence. However,. expression of E0240 is higher than that in I20260 because the pS1C3 is a high copy plasmid, while the pSB3K3 is low. J23115.E0240 was supposed to produce a fluorescent signal, too. But J23115 is weaker than J23101 considering a mutation existed. Therefore, the fluorescence signal was expected to be stronger in J23101.E0240 than J23101.E0240.

Results

Discussion

Attempting to exclude the interference of OD, we dilute the samples with LB medium in a ratio of 1:9. Compared with other data, this one shows superiority in linearity. So, considering the influence of bacteria density, we decide to use method above to accomplish the experiment.