Design

Foreword

Nowadays industrial development is changing rapidly, but the environment is sometimes sacrificed. Many kinds of pollution have been produced over the past few decades. People are now becoming more aware of environmental problems, and are attaching more importance to their health, so dealing with all kinds of pollution is necessary and urgent.

Water is the source of life, which means that the management of water pollution is very important. Heavy metal in the effluent plays a big role of water pollution, and we want to find out how to deal with nickel ions via synthetic biology. Last year our team-built Nickel Hunter 2.0 and had an idea of using S. Cerevisiae to absorb nickel ions, and this year we aim to work it out.

S. Cerevisiae system

S. Cerevisiae was chosen for two reasons. It has a strong environmental tolerance and was widely used to deal with all kinds of environmental problems. We came up with two parallel paths. One is to cause the yeast to transfer the nickel ions inside the cell, and the other is to get it to grab the nickel ions on the cell surface. By searching through the literature, we found three proteins that could meet our requirement.

First, we got the idea to reverse the application of protein purification (His-Tag), and instead use Hexa-his to attract and bind nickel ions to the cell surface. We were confident because scientists have previously used the α-agglutinin method to build a surface display system on the yeast.

We used MFα1+hexa-his+ AGα1 to realize our idea. MFα1 can express a signal peptide that guides the fusion protein to the outside surface of the cell membrane, hexa-his and AGα1 together can express the Hexa-his—α-agglutinin fusion protein. It has a GPI anchor at the bottom that can attach the C-terminal of the α-agglutinin to the cell wall, and the Hexa-his will be placed on the N-terminal.(Kuroda et al., 2001; Kuroda et al., 2002; Kuroda and Ueda, 2003)

The reason we want to use the surface display system is that it has two advantages. Its function is only affected by the amount of protein expressed and it can bind nickel ions even after the yeast is dead.

For the other path, we found two proteins, NixA and TgMTP1t2. NixA is a channel protein that can transfer nickel ions from the external environment into the cell’s internal environment(Deng et al.,2013), and TgMTP1t2 is a channel protein that can transfer nickel ions from the cell’s internal environment to the vacuole. Because the nickel ions will do harm to the yeast if they remain in the internal environment, we want to move them into the vacuole(Persans et al., 2001) which can safely store more nickel ions.

Thanks to the HZAU team for their proposal to use this protein by modeling.

Immobilized Yeast

We hope to control the location of yeast in the device and prevent gene leakage, the embedding method to do yeast immobilization is a good solution to this problem. Our raw material formula is 1g sodium alginate, 2g silica and 10%PVA liquid.

According to the literature and the experiments we have done, this method of fixing yeast allows yeast to maintain cell activity in very low pH and adding PVA makes the mechanical properties of gel balls better.(Liang and Huang,2009)

Device

This year, we built a mini-plant model of the effluent factory through 3D printing technology. It demonstrates our idea of applying engineered yeast to the process.

Because the engineered yeast must not escape into the natural environment, we employed gel balls to immobilize the yeast in order to easily to separate it from the effluent after treatment.

We also built software to automatically control the ingress and egress of effluent. We set a water level detector in the treatment pool to calculate the amount of effluent to be treated through the water level. The known volume of effluent can then be mathematically combined with our modeling, and thus the amount of yeast gel balls needed can be calculated.

Recycling

Solve local problems and put forward better optimization plan. We anticipated that the nickel ions absorbed can be released by the broken yeast, and the nickel elemental substance can be obtained through nickel electrolysis. This makes our project more complete, comprehensive and considerate.

References:

[1]Kuroda K , Shibasaki S , Ueda M , et al. Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions[J]. Applied Microbiology & Biotechnology, 2001, 57(5-6):697-701.

[2]Kuroda K, Ueda M, Shibasaki S, et al. Cell surface-engineered yeast with ability to bind, and self-aggregate in response to, copper ion[J]. Applied microbiology and biotechnology, 2002, 59(2-3): 259-264.

[3]Kuroda K , Ueda M . Bioadsorption of cadmium ion by cell surface-engineered yeasts displaying metallothionein and hexa-His[J]. Applied Microbiology and Biotechnology, 2003, 63(2):182-186.

[4]Deng, X., He, J., & He, N. (2013). Comparative study on Ni2+-affinity transport of nickel/cobalt permeases (NiCoTs) and the potential of recombinant Escherichia coli for Ni2+ bioaccumulation. Bioresource technology, 130, 69-74.

[5] Persans, M. W., Nieman, K., & Salt, D. E. (2001). Functional activity and role of cation-efflux family members in Ni hyperaccumulation in Thlaspi goesingense. Proceedings of the National Academy of Sciences, 98(17), 9995-10000.

[6]Liang xue, Zuxin Huang. Optimization of the Technology of S. Cerevisiae Immobilization by Sodium Alginate and PVA[J]. Liquor Making Science & Technology, 2009(2):27-30