iGEM 2022 — Team lzu-hs-china-e


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* Design Stage
* Build Stage
* Test Stage
* Learn Stage

Design Stage

![](https://static.igem.wiki/teams/4512/wiki/es/k1.png)

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.

Build Stage

1. Obtain the target gene fragment (the target gene fragment connecting InP, EC20, GroESL and IrrE)

2. PCR target gene

3. Preparation of carrier

4. Enzyme digestion vector and target gene

5. Connecting the target gene with the vector

6. Transformation of recombinant plasmid

7. Cell proliferation

Test Stage

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.

Growth under Salt Stress

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.

At 0% salinity, the control group grew fastest Because it added nothing and has the least burden. Engineering bacteria with foreign genes such as T7 and GroESL will have more burdens and make their growth more difficult. In this case, the control group with a small burden will have better advantages.

![](https://static.igem.wiki/teams/4512/wiki/es/3-1.jpg)

When the concentration of salt increases to 1%, the overall growth rate will exceed the situation when the salinity is 0%. Because an appropriate salt concentration can increase the growth rate of bacteria. But the control group is still the fastest since there is no high salinity to affect the growth of bacteria, so its additional genes still have no effect. These salinities are acceptable to bacteria themselves, and more genes still add a burden to them.

![](https://static.igem.wiki/teams/4512/wiki/es/3-2.jpg)

When the concentration of salt reaches 5%, the overall growth rate will be affected and drop a lot. After that, the role of GroESL and global regulators appeared. It exceede the control group and T7 with the highest growth efficiency.

![](https://static.igem.wiki/teams/4512/wiki/es/3-3.jpg)

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.

Growth of colonies

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.

Z has always formed the fewest colonies among the three strains, because it has not only inserted the gro promoter and the IrrE fragment for improving salinity tolerance but also inserted the EC20 and InP fragments for adsorbing heavy metals, which makes it too burdensome to grow as fast as other strains.

![](https://static.igem.wiki/teams/4512/wiki/es/3-4.jpg)

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.

Verification of heavy metal adsorption capacity

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.

In the bar graph, the vertical axis represents the adsorption capacity of EC20, and the horizontal axis represents the concentration of bivalent cadmium. The adsorption capacity of R-p strain and R-E strain increased with the increase of the concentration of bivalent cadmium. The adsorption capacity of the strains represented by R-E was stronger than that of the strains represented by R-p from the beginning.

![](https://static.igem.wiki/teams/4512/wiki/es/3-5.jpg)

Learn Stage

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.

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