Team:FAFU-CHINA/Design









Design


Ideas

For Human Practice, we found out that activities such as unpreceded use of agrochemicals and long-term application of urban sewage sludge, industrial waste disposal, waste incineration are the main sources of heavy metal in agricultural soil. While remediation of heavy metal in contaminated soil is done by physical and chemical processes which are costly and non-sustainable. In this case, we planned to combat heavy metal pollution problem in the soil through the alliance between microbe and plant, by manufacturing our chassis——Bacillus megateriumthrough getting the different complement of metal-binding proteins that handle metals inside cell as a metal-trap to alleviate metal stress of phytoremediation.





Metal-binding proteins

Our alleviating system refers to two kinds of metal binding proteins-MT and MBP derived from 2014 Cornell(BBa_K2384007) and 2010 Peking University Biobricks(BBa_K346003,BBa_K346004) respectively.

MTs(metallothioneins)-are low-molecular weight, cysteine-rich metal-binding proteins found in wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs contain 61-62 amino acids and are structually composed of two globular metal-binding domains(α and β) with metal ion. The C-terminal α domain contains 11 cysteines and the N-terminal β domain contains 9 cysteines. The 20 cysteine residues in MTs allow it to bind up to a total of 7 cadmium ions.[1]Several systems have been developed to allow for stable construction and high level expression of recombinant proteins in bacteria. To avoid being degradated by proteolytic enzyme, the expression of MT in frame with GST allows for stable expression. Im previous research, the fusion protein was prone to increase metal ion sequestration in E.coli, this time we intended to try to express fusion protein in our chassis.

This figure is engineered metal binding domain of MerR family.( Protein structure constructed by BNU)

MBP(engineered from PbrR and MerR respectively)-PbrR and MerR are members of the MerR family of metal-sensing regulatory protein, acts a effective Pb(II) and Hg (II) capturer respectively. According to earlier research, the DNA binding domain and metal binding domain can function individually and the constructed peptide can form a stable dimer with its mercury and lead binding affinity remaining. In order to reduce side effects of over-expression, Peking University tandemed two copies of metal binding domain of MerR and PbrR.

This figure is 3D structure of GST-MT.( Protein structure constructed by BNU)

Previous research showed that different localizations affect metal biosorption capability. [2]In this regard, MBPs were expressed in either the periplasmic and outer membrane by using DsbA and Lpp-Omp fusion partner respectively.

Xylose-inducible expression system: strong PxylA promoter and repressor gene xylR; after xylose addition, the repressor is released from the PxylA that activates transcription initiation.They have been designed for high expression in B. megaterium.





ACC deaminase

High levels of ethylene, produced under stressed conditions, can inhibit certain processes such as root elongation. 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into α-ketobutyrate and ammonia,simultaneously enhances plant growth and biomass by P solubilization and uptake particularly under stress condition by heavy metals. In our project, we aimed for increasing ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate heavy metals.






Summary

We hope to establish a sustainable, regulable and reusable project to solve the soil polluted by heavy metals. Phosphate-solubilizing bacteria have important functions. In our project, we used the mechanism of the alliance between microbe and plant, by manufacturing Bacillus megaterium, which is a kind of phosphate-solubilizing microorganism exists in the root system, forcing it enhance the plant remediation from two aspects, accumulating heavy metals and defend adversity stress. To achieve the goal of spatial specificity, we make most of the expression system which is regulated by root organic acid. We also used MBP (metal binding protein) and ACC deaminase to make accumulation of heavy metal in root system’s soil success. Finally, heavy metals will transfer to plant itself. A series of transformation we made in phosphate-solubilizing bacteria in our project will solve the weakness of hyper-accumulators, therefore, the remediation method will be put into use widely.





reference

[1]Haq, F., Mahoney, M., & Koropatnick, J. (2003). Signaling events for metallothionein induction, 533, 211–226.

[2]Kao, W., Chiu, Y., Chang, C., & Chang, J. (2006). Localization Effect on the Metal Biosorption Capability of Recombinant Mammalian and Fish Metallothioneins in Escherichia coli, 1256–1264.







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