Project background

1.1 High salt wastewater

High salt wastewater usually refers to wastewater containing NaCl 30g/L. Wastewater contains a large number of heavy metal ions, especially divalent cadmium will seriously affect our life and health. Due to its complex and diverse composition, high salinity, and strong inhibition of microbial growth, the technical difficulty of this wastewater treatment is much greater than that of ordinary wastewater treatment. Because the salinity in wastewater is far from the salt tolerance of ultramicroorganisms, it is extremely difficult for microorganisms to survive and reproduce in such wastewater. Cadmium is widely used in various industries in our industrial production. When cadmium enters the body, it accumulates in key organs such as the liver and kidney. "The kidney, which can accumulate and absorb up to 1/3 of the total amount, is the main organ of cadmium poisoning. The International Agency for Research on Cancer classifies it as a Group 1 carcinogen, and the Environmental Protection Agency classifies it as a Group B1 carcinogen.

1.2 cadmium hazards

Rice is a well-known aggregator of divalent cadmium. Cadmium is enriched in rice. Cause chronic poisoning of organisms. The chronic toxicity of cadmium usually begins with renal dysfunction, followed by bone dysfunction. Cd(II) -containing wastewater is highly toxic, and Cd(II) -containing compounds are more toxic. Cdcl2, for example, is lethal at very low concentrations. Because cadmium is a common industrial material, the soil around mining areas is often heavily polluted by heavy metals. As the current passes, it carries these sediments into the water, wreaking havoc on aquatic life

Solution

2.1 Chemical Methods

Methods in chemistry are generally divided into chemical precipitation, electrochemical techniques, and REDOX methods. This method usually involves the removal of heavy metal ions from wastewater by reaction with chemical reagents. However, the chemical method can not complete the treatment at one time, and a second treatment is needed.

2.2 Physical Methods

Secondly, on the physical level, there are adsorption, ion exchange, and membrane separation. This kind of method does not have the chemical reaction to carry on the ion adsorption, that is, uses the material basic nature to remove the heavy metal ion.

2.3 Biological Methods

Finally, the biological aspects of the method are mainly to use animals and plants, microorganisms, and other organisms through flocculation, enrichment, and other action to treat heavy metal ions in wastewater. The other biological method used by our team is activated sludge. The activated sludge method is to remove heavy metal ions from water by complexing, adsorption, and ion exchange of coordination groups contained in extracellular polymers of microorganisms with heavy metal ions. However, the general organisms have poor tolerance to the high concentration of salt, and the nutrients in high-salt wastewater are not sufficient, which leads to the poor treatment effect of the biological method on high-salt wastewater containing heavy metal ions. So, we're going to use engineered bacteria after biofortification to make it less restrictive.

Design of engineering

bacteria

3.1 Heavy metal adsorption gene

The cell wall of microbial cells plays a key role in removing metal ions from aqueous solutions due to the presence of a large number of functional groups with different charges and geometry that can interact with heavy metal ions. So we need to give it a gene that can adsorb heavy metal ions.

3.1.1 EC20 gene

EC20 is a qualified gene that can provide engineered bacteria with the ability to adsorb heavy metals. Cd (II) biosorption can be enhanced by displaying EC20 on the surface of Escherichia coli. We used a display system to fuse the EC20 protein to the surface of Escherichia coli cells, which is the N-terminal region of ice nucleating protein that serves as a cell surface display motif. But EC20 is only expressed inside cells, which does not help the cells adsorb heavy metals around them. Therefore, it is necessary to use cell surface display technology to make EC20 can be displayed on the surface of the cell to achieve the effect of adsorption of surrounding heavy metals.

3.1.2 INP gene

One such gene is INP, which can increase cell affinity and adhesion by anchoring on cells. Ice nucleating protein (INP) is used as a cellular anchor motif protein for surface expression, anchored to the outer membrane due to the presence of INP, and is stably expressed in the outer membrane. The water then fuses INP with EC20 to extend the heavy metal ions to the cell surface. That is, INP is used to anchor on the cell surface, heavy metal ions are adsorbed to the cell, and then EC20 is used for adsorption treatment inside the cell.

3.2 Salt resistance genes

Now the problem of heavy metal ion adsorption by engineered bacteria is solved. However, our goal in engineering bacteria is to make a microorganism that can still remove heavy metal ions under a high-pressure environment, so we need to provide a high salt tolerance gene to the cell.

3.2.1 IrrE gene with groESL promotor

IrrE can positively regulate salinity and drought tolerance through various stress response pathways, and its heterologous expression can significantly improve host stress tolerance. From these aspects, we can determine that the global regulator is a problem that can help cells from various aspects to fundamentally solve the problem of salt tolerance. Here we use the global regulator IrrE to give salt resistance that can survive in high-salt wastewater. IrrE is a global regulatory protein found in S. radiodurans. It improves the resistance of S. radiodurans to extreme stress environments by repairing DNA damage under stress conditions such as strong radiation. Therefore, as a transcription factor in microorganisms in an extreme environment, gene participated in the process of tolerance response of rape seedlings to NaCl stress through heterologous expression, and directly or indirectly regulated the expression of genes related to NaCl stress response, thus improving the tolerance ability of plants to NaCl stress. This is achieved with the help of the groESL promoter, which increases its stress resistance and allows it to survive in highly saline wastewater. groESL promoter plays the role of initiating IrrE and helping IrrE to improve the salt tolerance of cells. (The salt tolerance of organisms results in their ability to survive in a range of salinities, and within K+ of their acceptable range, organisms can function normally. However, beyond their acceptable range of K+ can lead to the folding of proteins in these organisms and the improper functioning of DNA and protein interactions in the cell, leading to the unhealthy and even death of the cell. The E. coli that we're working with doesn't have enough salt resistance to survive the high salinity of the wastewater and needs some outside help to survive the high salinity of the wastewater. So global regulation of genes comes into play. First of all, there are two ways to make a gene more salt tolerant. It can confer salt tolerance through functional genes as well as regulatory genes.

First, functional genes are a way to help cells survive through transporters that maintain ion homeostasis, enzymes that synthesize various osmotic protectors, and detoxifying proteins that directly protect functional cell proteins. However, stress tolerance is a complex mechanism that needs to be controlled by multi-layer regulatory networks. The use of regulatory factors helps regulate the whole cellular process of microorganisms, but cannot be achieved by overexpression of single functional genes. As a result, this method is not a good solution to the existing problems. Thus, we are left with the solution of global regulatory genes, which are regulators that help gene expression, regulators involved in various stress conditions, signal transduction proteins, and protein kinases. This can make better use of the introduction of a single foreign gene to improve the stress tolerance of microorganisms.)

3.3 Overall conclusion

In the conclusion, INP was used to anchor on the cell surface to adsorb heavy metal ions to the cell, and then EC20 was used to adsorb the inside of the cell. By using the expression of the cell, the heavy metal ions can be absorbed by it.

IrrE gives salt resistance that can survive in highly saline wastewater. This is achieved with the help of the groESL promoter, which increases its stress resistance and allows it to survive in highly saline wastewater.

Project inspiration

The increasingly serious cadmium pollution has brought a great influence on our life. Water sources are polluted, posing a great threat to aquatic life. The water flows through the farmland, bringing divalent cadmium into the wheat, and cadmium-containing rice into the market, which can damage the kidneys and liver and cause "Tongtong disease" when eaten by citizens. Many people have been harmed by it. Because of the salinity intolerance of microorganisms, cadmium treatment is extremely difficult, so the problem has been waiting to be solved. We came up with the idea of a gene that can survive in high-salt wastewater and absorb heavy metal ions to solve this problem.