EC20 is a qualified gene that can provide engineering bacteria with the ability to adsorb heavy metals. Biosorption of cd(II) can be enhanced by displaying EC20 on the surface of E. coli. But EC20 is only expressed inside the cells, which does not help the cells to absorb the surrounding heavy metals. Therefore, it is necessary to use cell surface display technology to allow EC20 to be displayed on the surface of cells so as to achieve the effect of adsorbing surrounding heavy metals.

INP is the gene that can increase cell affinity and adsorption by anchoring to cells. The extension of heavy metal ions to the cell surface can be achieved by water fusion of INP and EC20. That is, INP is used to anchor on the cell surface, heavy metal ions are adsorbed to the cells, and then EC20 is used for adsorption treatment inside the cells.

Now the problem of engineering bacteria adsorbing heavy metal ions has been solved. However, the goal of our engineering bacteria is to enable microorganisms to remove heavy metal ions in a high- stress environment, so we need to provide a high salt tolerance gene to the cells.

Global regulators are one that can help cells in various ways to fundamentally solve the problem of salt tolerance. Here we confer salinity resistance that can survive in hypersaline wastewater through the global regulator IrrE. This effect is achieved with the help of the groESL promoter, increasing its resistance to stress and allowing it to survive in high-salt wastewater. The groESL promoter plays a role in initiating IrrE, helping IrrE to improve the salt tolerance of cells.

At this stage, we make a comparison so that we can more intuitively show what changes the added genes have made to our engineered bacteria after we've successfully built it.

We compare three different groups of strains. One group is the control group, DH5alpha, the strain that has not been genetically modified. The second group added T7 promoter. The third group is the final product of our experiment, which adds the complete sequence, that is, the ire gene with the GroESL promoter to help its gene expression. We tested the growth in different salt concentrations.

In the end, when the salt content reaches 7%, the growth efficiency of all three groups will be almost zero because of the too high salt concentration, which means that it is almost impossible for them to grow at this time because it has become very difficult for them to survive under that condition.

We verify the number of colonies formed by different strains under different salt concentrations. Z represents the insertion of InP, EC20, gro promoter and IrrE global regulators to increase salt tolerance and heavy metal adsorption capacity, gro represents the insertion of only gro promoter and IrrE global regulators. C represents the control group without any insertion.

The strain represented by gro has always been the fastest growing strain because it only inserts the gene fragments of the gro promoter and the ire global regulator, which does not make its growth burden heavier, and the insertion of the ire global regulator improves its salt tolerance so that it can grow better in saline.

R-p represents strains with InP, EC20 and gro gene fragments inserted. R-E represents strains with IRR gene fragments inserted in addition to InP, EC20 and groESL.

In the broken line graph, the horizontal axis represents the concentration of bivalent cadmium, and the vertical axis represents the percentage of bivalent cadmium absorbed. In the figure, the percentage of bivalent cadmium absorbed by the strains represented by R-p is always much less than that represented by R-e.

Although this experiment has a successful result, there is still a lot of room for improvement in the experimental design, which can make the next experiment have a result with higher value, more practicality and application. Selection, genetic design, etc.

Let’s talk about the selection of strains first. The strains we need have to be easy to cultivate, highly common, have low editing difficulty, and have strong adaptability to harsh environments. So we used E. coli, but there are still more strains to choose from. While E. coli is a very common strain, and it's true that it's not that difficult to achieve gene editing, there seem to be other, better options out there. It is not entirely because of the advantages of E. coli, but only E. coli is selected. We can try some strains with higher salt tolerance or faster reproduction in sewage environments or even stronger living ability in harsh environments. There are still many choices, not only limited to E. coli strains, but also other better choices to achieve better efficiency and effect.

Furthermore, for the gene design, we selected four genes, Inp, EC20, grESL and IrrE, to achieve sufficient salt tolerance and cadmium adsorption, and to maintain proper fertility and tolerance in a high-salt environment. To ensure that our strains can survive in this environment and achieve the amount of cadmium we need to adsorb. For anchoring, there is not only the option of inp, but you can choose to anchor more stable or display more efficient gene fragments. For EC20, we can choose the genetic design with stronger adsorption to heavy metals or stronger tolerance to heavy metal toxicity. Finally, there is irrE which is a globally regulated gene for the regulation of salt tolerance. But there are still other genetic designs that are more regulated, or more resilient in high-salt environments, with minimal impact on reproduction speed.