Design

# Design

## 1.transportation——chelate——redox

Cadmium contamination of water worldwide is already having a severe environmental impact, especially on crops, such as cadmium rice, and in addition to this, cadmium contamination is likely to cause itai-itai disease and renal tubular injury, after prolonged and/or high exposure may eventually to renal failure, thus, the problem of cadmium pollution of water needs to be solved urgently.We adopt an environmentally friendly biological approach——use Synechocystis sp. PCC6803 with a clear genetic background and a certain degree of tolerance to cadmium as a chassis organism for transformation.

First of all, we chelate cadmium, which is enriched on the cell wall and efficiently absorbed into the cells through transporter proteins. However, which may cause photosynthesis failure, protein inactivation or oxidative stress in cyanobacteria and lead to cell death,making it difficult to achieve the high tolerance rate. Therefore, after transporting large amounts of cadmium ions into the cells, we can accumulate heavy metal ions and prevent them from being transported out of the cells by means of chelating proteins, and at the same time increase the expression of oxidoreductase to remove reactive oxygen clusters in the cells, thus increasing the growth rate of algae under cadmium ion stress. Besides, for high expression of functional proteins, We optimized the codon preferences of the algae, and analyzed the protein expression results.(Figure 1)

Figure1: The core “transport-chelation-redox reaction” system of our project

1.1Cd transporter

Although the cytoderm of algae has a strong ability to adsorb heavy metals, but when used in wastewater treatment, the removal rate is still unsatisfactory, and extracellular adsorption is not very stable, so we need one or more cadmium transporter that can transport cadmium from the environment into the cell in an efficient and controlled manner. These transport proteins need to meet the following characteristics.

1. It functions efficiently in cyanobacteria. Since proteins expressed in eukaryotes are highly efficient and less susceptible to the cellular background environment, we chose IRT1 and OsNRAMP5, which are highly conserved. Considering the risk of low expression of eukaryotic proteins in prokaryotic cells, we also selected the MntH protein that was well expressed in Bacillus subtilis by the Newcastle team in 2009.
2. High translocation efficiency. Since it is very difficult to measure the transport efficiency of the protein directly, we measured the transport efficiency of the protein indirectly by the effect on the cellular efficiency of cadmium uptake when the protein was heterologously expressed in previous experiments.
3. Strong specificity. Cd transporters usually transport large amounts of Ca²+, Zn²+ and other divalent ions, if other ions are flooded into the cell without being processed, it is easy to cause intracellular ion imbalance. We can choose the protein with good specificity by the position of expression in the cell and the possible function of the protein.

Introduction of selected proteins

1.1.1 OsNRAMP5

It is a heavy medal transporter expressed mainly on roots and can take up $$Cd$$ into cells. Experiments in plants show that mutated with Carbon Ion Beams, the unmutated parental $$Cd$$ content was 1.73 mg $$Cd⋅kg^{-1}$$, and $$<\ 0.05 mg\ Cd⋅kg^{-1}$$ in the daughter grain after mutation. In addition, this family has similar sequences in prokaryotes.

1.1.2 MntH

2009 Team Newcastle（http://2009.igem.org/Team:Newcastle/Project）expressed it on Bacillus thuringiensis using it to take up cadmium ions. Bacillus subtilis also belongs to the NRAMP family, which is a proton-coupled metal ion transporter.

1.1.3 IRT1

IRT1 is an Fe ion transporter, which is located on the plasma membrane and can transport metal ions from roots to epidermal cells under the condition of iron deficiency. Studies have shown that IRT1 may be a key gene for Cd uptake in amaranth, and Fe/Zn deficiency can promote the expression of iron ion transport protein IRT1, and overexpression of IRT1 can increase the accumulation of Cd uptake in plants.

1.2 Chelate protein

Under high cadmium stress, cells express a large number of chelating proteins to chelate cadmium ions for abatement, with the best effect of metallothionein(MT) (cited) being highly conserved in various species and more likely to be expressed in our chassis organism.Metallothioneins are a class of small-molecule proteins with low relative molecular masses and rich in cysteine, which can take up Cd and chelate to the sulfhydryl group of proteins in an inactive form.According to the different expression sites of metallothionein, it can be divided into membrane expression and cytoplasmic expression. Metallothionein expressed on the membrane can adsorb cadmium ions outside the bacteria and enrich them on the bacterial cell membrane, which is conducive to the transport of transporters; metallothionein expressed in the cytoplasm can chelate cadmium ions and form complexes with them to reduce the toxicity brought by high concentrations of cadmium ions.

Introduction of selected proteins

1.2.1 SmtA (on the cell wall)

Itself comes from the Synechococccus (Synechococcus PCC 7942), which is expressed on the cell surface, adsorbs heavy metal ions, and upon expression is 2.8 times more likely to be absorbed by E. coli without SmtA [1]. We used SmtA expression on the cell wall of the cytobacterium Synechocystis to enrich cadmium ions in the waters and to increase the efficiency of the transport protein.

1.2.2 hMT-1A (in the cell)

The efficiency of intracellular accumulation of Cd was 6.36 mg Cd/g dry cells [2]hMT-1A contains two domains Domain, α domain and β domain, one α domain can bind 3 Cd and one β domain can bind 4 Cd. We expressed it in the cytoplasm to chelate and collect Cd ions inhaled by the alga.

1.2.3 TMCd1 (in the cell)

Literature showed that the absorption capacity of recombinant strain to Cd increased to 7. 90 μ mol / mg dry cell weight, which was nearly 19 times higher than that of the control strain.

TMCd1 is made up of 156 amino acids, of which 47 are cysteine 2+ residues that can bind 16 Cd. Therefore, Suleman et al. expressed the TMCd1 gene fused to the glutathione S-transferase gene in E. coli BL21 DE3 cells. We used it in the cytoplasm to chelate the cadmium ions inhaled from the cytosol of E. coli.

1.3 Redox protein

The effects of heavy metal enrichment in the intracellular compartment are threefold: inactivation by binding intracellular proteins, attack on antioxidant enzymes and membrane systems, and destruction of genetic material.Our oxygen reducing proteins protect protein activity, photosynthetic activity, and membrane activity, respectively, to ensure the normal functioning of the whole cell under cadmium stress.There is also a regulatory relationship between lox1 and apx2, which can further enhance the expression level of apx2 by overexpression of lox1. As the photosynthetic system is crucial for the functioning of the algae, the expression level of apx2 is very important.

Introduction of selected proteins

1.3.1 APX2

The expression level of APX2 in Arabidopsis cells increases in response to cadmium stress. Overexpression of APX2 significantly decreases oxidative stress in cadmium-stressed cells. Molecular mechanism studies have shown that it can effectively protect the balance of photosynthetic system by eliminating the disorder of electron transport chain in photosynthesis under cadmium stress.

1.3.2 LOX1

LOX1 is an upstream regulatory molecule of apx2 and is able to significantly increase the level of apx2 by overexpressing lox1.LOx1 is also directly involved in the regulation of ROS and oxygen homeostasis of the membrane system as an antioxidant stress protein.

1.3.3 Cu/Zn-SOD

Superoxide dismutase (SOD) is widely found in aerobic organisms, anaerobic organisms and some anaerobic microorganisms, Superoxide dismutase (SOD) is widely found in aerobic organisms, anaerobic organisms and some anaerobic microorganisms, Cu/Zn-SOD can maintain high activity under high intracellular cadmium ions, and can effectively protect the cellular oxygen balance by eliminating free radicals produced by cadmium stress, regulate the intracellular ROS stress level and maintain a steady state.

## 2. Toxin-antitoxin" light-controlled suicide system

The escape of modified engineered algae is one of the major hazards of genetically modified organism, and the escape of our engineered Synechocystis sp.PCC6803 into the natural world may also lead to different adverse effects such as water bloom and reduction of water polymorphism. Based on these facts, we designed our blue-light-controlled "toxin-antitoxin" suicide system. (Figure 2) In this system we use the RelE/RelB-toxin/antitoxin system and regulate the expression of the toxin RelE through the blue light-regulated promoter[4]. In the presence of blue light, the regularly expressed HTH-LOV-REP blue-regulated protein will homodimerize and specifically recognize the (c120)5 sequence upstream of the blue-regulated promoter via HTH, while inhibiting the expression of toxin RelE via the REP domain[5]. When the algae escapes from the blue light environment we have set up, the uninhibited blue light regulatory promoter will initiate the expression of RelE and under the effect of our amplification system the content of RelE will increase rapidly, as a result the escaped algae will be killed. Meanwhile in order to prevent the small amount of RelE that is still expressed in the presence of blue light from harming the algae, we regularly express the antitoxin RelB to neutralize its toxin effect and ensure that the toxin RelE does not harm the unescaped Synechocystis sp.PCC6803.

Figure 2:Third-generation system the Blue light regulated suicide system

To sum up, these two systems (Figure2) enabled us to achieve the goals of the project.

Figure3:The whole system of our project.

## 3.Recycling of Ca-Alg MBs

We confine algae in Calcium-Alginate Microbeads，not only can immobilize our biological chassis, but also improve the cadmium elimination. After that we continuously expose them to blue light in a tube. In order to make our project more complete, we considered the recycling of cadmium, if the salvaged Calcium-Alginate Microbeads (Ca-Alg MBs) directly incinerated, the resulting cadmium exhaust will become secondary pollution, which is “putting the cart before the horse”, so our design is: after removing the blue light, the algae will die, cadmium ions will be part of the residue in the Ca-Alg MBs, so we will concentrate the balls in a confined device, add the right amount of sodium citrate hot solution to dissolve the Ca-Alg MBs, and finally the closed device will be handed over to the cadmium treatment plant for reasonable recycling.

## References:

1. Saffar, B., Mehri Ghahfarrokhi, A., Mahnam, K., & Mobini-Dehkordi, M. (2015). Improvement of Cd2+uptake ability of SmtA protein by Lys/Cys mutation; experimental and theoretical studies. Journal of Biomolecular Structure and Dynamics, 33(11), 2347–2359. doi:10.1080/07391102.2015.1054431
2. Ma, Y., Lin, J., Zhang, C., Ren, Y., & Lin, J. (2011). Cd(II) and As(III) bioaccumulation by recombinant Escherichia coli expressing oligomeric human metallothioneins. Journal of Hazardous Materials, 185(2-3), 1605–1608. doi:10.1016/j.jhazmat.2010.10.051
3. Suleman A, Shakoori AR. Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1 (TMCd1) gene in Escherichia coli. J Cell Biochem. 2012;113(5):1616–1622. doi:10.1002/jcb.24030
4. Harms, A., et al., Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology. Molecular Cell, 2018. 70(5): p. 768-784.
5. Ochoa-Fernandez, R., et al., Optogenetic control of gene expression in plants in the presence of ambient white light. Nat Methods, 2020. 17(7): p. 717-725.