Team:HBUT-China/Design

Design



On this page, we describe our design work in detail to show how Nickel Hunter 2.0 detects nickel ions, and how to apply it in real life.


Foreword

Electronic products have been widely used in our lives; unfortunately they have also brought about a new kind of pollution in the 21st century. More and more heavy metal ions, including nickel ions, are discharged into the environment due to improper handling. Based on this situation, Nickel Hunter 2.0 proposed by the HBUT-China team provides a new method for nickel ion detection via synthetic biology.

To achieve this goal, we designed a nickel ion detection/measurement system assembled in E. coli. It is based on the nikABCDE gene, which transports nickel ions in the environment into the cells, and the luxCDABE gene, which expresses bioluminescent proteins in the presence of nickel ions, thus reflecting their concentrations via luminescent intensity.


E. coli system

We found that the protein ncrB can bind to the promoter pncrA, resulting in the downstream gene not being transcribed. However, when a nickel ion binds to the protein ncrB, the inhibitionof pncrA can be eliminated[1]. Therefore, we want to use the above relationship between ncrB and pncrA to design a device that can detect the concentration of nickel ions. At the same time, we found that the nikABCDE system present in other strains of E. coli belongs to the ATP-binding cassette (ABC) protein family, and contains five proteins that can transport the hydrolyzed ATP to the transmembrane transport of nickel ions. NikB and NikC are two transmembrane proteins that form the transmembrane core of the transport system. NikA is a periplasmic binding protein (PBP) that transmits captured nickel ions to the NikBC core. NikD and NikE act as two cytoplasmic proteins[2]. The signal-to-noise ratio of the system is increased by the nickel ion channel protein, and the detection accuracy is improved. Furthermore, we found that LuxCDABE bioluminescent protein is capable of bioluminescence[3] , and can be used as our reporter gene. Compared to the previously identified mRFP reporter gene, it is self-luminous and does not require external light source excitation. In addition, it solves the problem of the naturally occuring flourescence in our E. coli which interfered with our measurement in last year’s project. When nickel ions in the environment enters the bacteria through the NikABCDE transporter, it binds with the protein NcrB, eliminating inhibition of the promoter pncrA, allowing it to express LuxCDABE fluorescent proteins, producing green fluorescence.


Machine

We will do this for our design products. We put the engineered bacteria containing the genetic line into the injection tray of the machine with tin foil paper. The injection tank has a thermostat device, which can ensure the temperature of the bacteria is not fluctuate in real time. Through chip processing, photoelectric conversion module and wireless module, it converts bioluminescence into digital signals and transmits them to our mobile phones for real-time monitoring[4].


S. Cerevisiae system

After successfully constructing the nickel ion detection system in E. coli, we envisioned the construction of a system that collects nickel ions. After reviewing the previous works and related contents of the literature, we have identified the biological chassis of this system as S. Cerevisiae. Yeast has a vacuole that is not found in E. coli, and the vacuole is an organelle that is isolated from the intracellular environment. Nickel ions are toxic to cells; however, if we store the ions in their vacuoles instead of their cell environment, we would greatly increase its resistance to nickel ions. This would make it a good candidate to achieve our purpose of capturing large amounts of nickel ions. We found a nickel ion transporter, TgMTP1t2, in a cell of a plant called Thlaspi goesingense hyperaccumulator, which is expressed in the vacuoles of these cells, which we subsequently successfully expressed on our S. cerevisiae cells. However, we thought that the capture of nickel ions by the yeast should be a gradual process, that is, first from the outside to the inside of the cell, and then from the cell environment into the vacuole[5] . So we hope to find a gene that can transport nickel ions from outside the cell into the cell. We found a nickel-ion transporter called TjZNT1 on the ultra-nickel-resistant plant called Thlaspi japonicum, which is a ZIP family located on the cell membrane[6]. According to the literature, this protein has been successfully expressed on S. cerevisiae cells and can achieve the purpose of transporting nickel ions[7]. So, as of now, we have found two nickel ion transporters and want to express them in S. cerevisiae for nickel ion capture purposes. Due to limited time, we have not built the relevant mechanism, but hopefully next year we can build this system.


Reference

1. Tao Zhu. Study on the Mechanism of Regulation for the Nickel Resistance Determinant Expression in Leptospirillum ferriphilum UBK03. Chinese Academy of Agricultural Sciences Dissertation. 2011

2. Dosanjh N S, Michel S L. Microbial nickel metalloregulation: NikRs for nickel ions[J]. Current Opinion in Chemical Biology, 2006, 10(2):123-130.

3. Winson M K, Swift S, Hill P J, et al. Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs.[J]. Fems Microbiology Letters, 1998, 163(2):193.．

4. Mimee M, Nadeau P, Hayward A, et al. An ingestible bacterial-electronic system to monitor gastrointestinal health[J]. Science, 2018, 360(6391):915.

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

6. Mizuno T, Usui K, Horie K, et al. Cloning of three ZIP/Nramp transporter genes from a Ni hyperaccumulator plant Thlaspi japonicum and their Ni2+-transport abilities[J]. Plant Physiology & Biochemistry, 2005, 43(8):793-801.

7. Mizuno T, Usui K S, Unno T, et al. Investigation of the basis for Ni tolerance conferred by the expression of TjZnt1 and TjZnt2 in yeast strains[J]. Plant Physiology & Biochemistry, 2007, 45(5):371-378.