Team:XMU-China/Project Interlab

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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.

 

Figure 1. GFP of different device under nature light. 1: BBa_K1412999 in DH5α; 2: BBa_K1412716 (reconstructed by us) in DH5α; 3: BBa_K1412716 in DH5α; 4: BBa_K1412924 in DH5α.

 

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.

 

Figure 2. GFP of different devices under the UV-light. 1: BBa_K1412924 in DH5α; 2: BBa_K1412716 (reconstructed by us) in DH5α; 3: BBa_K1412716 in DH5α; 4: BBa_K1412999 in DH5α.

 

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




1.1kb Marker;

2.Double restriction enzyme digestion (EcoR I and Pst I) with device BBa_J23115 + BBa_E0240 (constructed by us).

3.Mono-restriction enzyme digestion (Pst I) with device BBa_J23115 + BBa_E0240 (constructed by XMU-China).

4.Double restriction enzyme digestion (EcoR I and Pst I) with device BBa_I20260 (reconstructed by us).

5.Mono-restriction enzyme digestion (Pst I) with device BBa_I20260 (reconstructed by us).

Figure 3. Enzyme digestion verification of devices BBa_J23115 + BBa_E0240 and BBa_I20260.

Results and discussion: For device BBa_I20260, it’s very abnormal that we get puzzling results with double enzymes digestion by Xba I and Pst I. The target segment seems vanished. However, if we use EcoR I and Pst I instead, we find that the segments, whose backbone is PSB3k3, generated by mono-restriction digestion is about 1000bp longer than that generated by double restriction enzyme digestion, and the devices have been verified by DNA sequencing. We can also get segments slightly shorter than 1000bp which generated by double digestion, and those segments are highlighted by red frames. So we confirm that device BBa_I20260 is correct. We took our actual measurement with the reconstructed device.








1.100 bp Marker.

2.BBa_K1412924 with double enzymes (Xba I and Pst I) digestion.

3.1kb Marker.


Figure 4. Enzyme digestion verification of devices BBa_J23101 + BBa_E0240

As double restriction enzyme digestion generates two target segments, we confirm that device BBa_J23101 + BBa_E0240 is constructed correctly.


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.

Figure 5. The plot of OD versus time. Top row are the plots of OD versus time for each single device. Bottom row are combination data of all three devices with error bar. We can conclude that the growth rate is getting slower with time increasing. We measured each device with three samples for three times parallelly, and we make sure that the reproducibility of the data is acceptable. Comparing the plot of OD versus time with each other, we can see that their growth rate are almost the same.


Figure 6. The plot of RFUs versus time. Top row are the plot of RFUs versus time for each single device. Bottom row are combination data of all three devices with error bar. From the plots, we can conclude that the RFUs increases linearly with time. Comparing with Figure 5, we can find that the GFP expression strength of all three devices from strong to weak is: BBa_K1412924, K1412716, K1412999. The relationship meets the assumption based on iGEM database. Because the activity of promoter J23101 is stronger than J23115, while the copies of backbone pSB1C3 is higher than pSB3K3. So that the result is reasonable.


Figure 7. The plot of RFUs versus OD600. Top row are the plot of RFUs versus OD600 for each single device. Bottom row are combination data of all three devices with error bar. From the plot, we can find that the RFUs increases linearly with OD600, and we can conclude that the GFP expression strength of all three devices from strong to weak is: BBa_K1412924, K1412716, K1412999.


Figure 8. The plots of RFUs/OD600 versus time for each single device. Top row are the plot of RFUs/OD600 versus time for each single device. From the plot of RFUs, we regard the RFUs/OD600 as a representation of the fluorescence expression strength of unit bacteria. So we can get that the GFP expression strength of all three devices from strong to weak is: BBa_K1412924, BBa_K1412716, BBa_K1412999.


Reference:

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

 

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