Team:XMU-China/Project Interlab

INTERLAB STUDY

The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. 

After the experiment, we get three parts:

BBa_K1412716: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 backbone.

BBa_K1412924: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), in the pSB1C3 backbone.

BBa_K1412999: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), in the pSB1C3 backbone.

 GFP generator with J23101, J23115

For devices BBa_I20260 and BBa_J23101 + BBa_E0240. Both devices consist of Anderson promoter J23101 and GFP generator BBa_E0240. When the device is constructed in backbone pSB3K3. A low copy number is in expectation, as a result, a weak fluorescence strength is shown. While the device is constructed in pSB1C3 which is a higher copy number vector, hence a stronger fluorescence strength, so that it can be obvious enough to be observed in naked eyes.

For device BBa_J23115 + BBa_E0240, the promoter strength is the weakest, so that it’s difficult to observe green color in naked eyes.

The bacteria was cultured in the LB medium for 12 hrs at 37℃ shaking at 200 rpm in the table concentrator, then 1 ml bacterium solution was transferred into 1.5 ml centrifuge tube, and was centrifuged at 10000 rcf(g) for 1 min. The supernatant was discarded and the residuals was suspend by PBS. The solution was centrifuged again, and we got the bacteria precipitate as the picture shown in Figure 1. In which we can find that device BBa_K1412924 is greenish in natural light while device BBa_K1412716 (reconstructed by us) and BBa_K1412716 emit a canary yellow color, and device BBa_K1412999 show the color which is close to white.

Under UV-light, the bacterium precipitate above can be observed clearly that device BBa_K1412924 can emit strong green fluorescence, while devices BBa_K1412716 (reconstructed by us) and BBa_K1412716 have weaker green fluorescence, and the green fluorescence from device BBa_K1412999 is the weakest so that we can’t even observe a green pixel.

Devices Verification

Protocol

1. Transformed BBa_K1412924 into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃.

2. Inoculate a 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate.

3. Cultures were grown in conical flask for 16 hrs at 37℃ with shaking at 200 rpm in the table concentrator.

4. Cultures were diluted 1:100 into three 20 ml fresh LB medium and grown for 3 hrs at 37℃ with shaking at 200 rpm in the table concentrator.

5. Then transfered 650 μl of the culture to a 1.5 ml centrifuge tube, centrifuged and washed twice with phosphate-buffered saline (PBS, pH 7.4) to minimize the background fluorescence from the medium.

6. The washed cells were suspended in PBS and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements.

7. Measure the fluorescence and absorbance:

(1)Fluorescence:

Device: SpectraMax+M5 microplate reader, 96-well plates.

Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.

(2)OD600 (optical density at 600 nm):

Device: SpectraMax+M5 microplate reader, 96-well plates.

Wavelengths: 600 nm absorption.

8. Measure every 30 minutes in the next 4 hrs.

Experimental data

Before the measurement, in order to set date in suitable range we estimated an appropriate concentration range by diluting the bacterium culture 2 times with PBS buffer. The values of PBS, the background, were subtracted during the data processing. And the data was removed when their deviations were too large. Then the remained data were doubled to get the final values to plot graph.

1.

Reference:

1. Bagh, Sangram, Mahuya Mandal, and David R. McMillen. "Minimal genetic device with multiple tunable functions." Physical Review E 82.2 (2010): 021911.