Team:HUST-China/Design

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Design

After verifying the adhesion of our surface displayed Si-tag and secreted Mcfp-3, we decide to stimulate the real working conditions to confirm our engineered strain’s ability to solve practical problems in the real environment.

Considering the real-world conditions, we urgently want to know whether our Euk.cement cell diffuse into the sands well? So firstly, we used modeling to stimulate the diffusion situation that how the cells move in the seabed. The modeling simplified some real situations reasonably. We hope to make our design of verification fit real world better, and can give guidance to the ultimate working plan in the real seabed environment.(Click HERE to see more details of our modeling)

The conclusions we got from the modeling:
-The Euk.Cement permeates very efficiently over a large space. Therefore, perhaps we have to enclose it with fence.
-the big particles such as rocks or other materials mixed in sands have little effect on the Euk.Cement permeation.
-The Euk.Cement distributed quite evenly in the whole space they spread but not densely at the surface of the sands.
With the support from modeling, it seems that we don’t need to worry so much about the diffusion. High diffusion efficiency benefits a lot in the real world situation. And we also paid much attention to the safety of project, so we prepared a container for our strains.

We finally started to the design of our special device.

It is obviously that the physical and chemical indicators, like the pH or temperature, will not fluctuate acutely in the sea.Therefore, we tried to assemble a device by the things we can easily get in biological lab to build a relatively stable environment of sandy seabed.We referred to a number of verification methods in the literature concerning microbial reinforcement and the microporous material commonly used measurement instruments in civil engineering.

Figure 1：Devices used in civil engineering

This realistic condition’s verification design combines both traditional biological experiments and the concept of Civil Engineering Experiment. Ultimately, we have a following device, DIY for our verification:

Figure 2：The principle of device that we designed

Figure 3：Devices used in our lab

We used 50ml glass syringe to simulate our operating environment, and put quartz sand mixture on the bottom of glass syringe. The upper of the syringe was filled with salt solution to simulate the real environment with salt stress.

The experiment was in room temperature, we sealed the top of glass syringe after pumping yeast culture into syringe. Referring to the papers, we replaced the culture every 3 hours for a cycle. (Click HERE to see more details :Verification Experiments -part 4)

Result:

Figure 4: Sands cementation with testee cells(Si-tag +Mcfp3). A: Sands cementation test was carried out in lab with trial column and quartz sands. B, C: Sands treated with testee cells (Si-tag+Mcfp3) form cementation in columns. We can see sands were stacked together by microscopy. D, E: Sands treated with wildtype control cells cannot form cementation.

In fact, the results really exceeded our expectations. Even under the salt stress, our marine yeast still performed excellently.After half of the processing time compared to reference, we got a complete consolidation result, a consolidated sand column. We believe our hard-working marine strains can survive and work for longer time.

We can proudly announce that our Euk.cement can indeed play a role in the real sandy seabed environment. And it has the ability to build artificial reefs with different intensity in a way totally different from the traditional one. Our design of Euk.cement is an environmental-friendly, energy- friendly biological reinforcement method, to resolve our concerns on environment.

Combining the modeling and the experiment result, we put forward a reasonable implementation method:
The engineered yeast must be stored in container with a constant light lamp (inhibition device). It can be transported to appointed place in a normal concrete truck in which sands and Euk.cement can be mixed in darkness and cementation is started in advance. Or we can also transport Euk.cement to appointed place with light, and then mixed with local sands in darkness to initiate system working. In either way, after about 60 hours mix in darkness, the container together with sands and working Euk.cement can be sink deep underwater to the target region that is sealed with fence. The sands mixed with Euk.cement are released. Cementation will finally be performed silently. Besides of fences, the Si-tag displayed on cell surface can further limit the diffusion of cells. So we can put the cementation under control.

Figure 5: a diagrammatic sketch for our implementation method

Throughout the design we can see that our engineering yeast does have the ability to work under realistic conditions. However, we also found some shortcomings, its bond strength still can’t replace the traditional chemical or mechanical methods. But this does not prevent our footsteps. Mechanical reinforcement needs a lot of energy, and the use of chemical reagents can easily pollute the environment. Nowadays, this traditional methods do is being widely used, but this environmental- unfriendly technology will be eliminated one day.

We believe that, in the near future, micro-consolidation based on synthetic biology will become the most valuable technology of human beings. It will help build a more wonderful world. And we, iGEMers are making great efforts on it!

References

[1] Nemati M, Voordouw G. Modification of porous media permeability, using calcium carbonate produced enzymatically in situ[J]. Enzyme and Microbial Technology, 2003, 33(6): 635-642.

[2] DeJong J T, Mortensen B M, Martinez B C, et al. Bio-mediated soil improvement[J]. Ecological Engineering, 2010, 36(2): 197-210.

[3] de Muynck W, Verbeken K, de Belie N, et al. Influence of urea and calcium dosage on the effectiveness of bacterially induced carbonate precipitation on limestone[J]. Ecological Engineering, 2010, 36(2): 99- 111.

[4] de Muynck W, de Brouwer D, de Belie N, et al. Bacterial carbonate precipitation improves the durability of cementitious materials [J]. Cemenet and Concrete Research, 2008, 38(7): 1005-1014.