Growth under Salt Stress

After we have successfully constructed our engineered bacteria, we need to compare its functions. At this stage, we need different groups to make a comparison so that we can more intuitively show what changes the added genes have made to our engineered bacteria.

Now we will compare three different groups of strains. One group is the control group. That is, DH5alpha, which has not been changed at all, has not let us insert any genes. That is, the strain that has not been genetically modified is used as the control group. 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. However, in the end, we will put them together and observe their growth under different salinity.

When the concentration was 0%, the control group grew the fastest at the same time. Because the control group that adds nothing at this time has the least burden. Engineering bacteria with foreign genes such as T7 and GroESL will have more burdens and make their growth more difficult.

Without these, the burden will be much smaller when growing, and this is under the condition of a salinity of 0. However, the growth of these strains is not very good, because certain salinity is also indispensable for the growth of strains. In this case, the control group with a small burden will have better advantages. But GroESL and ire with salt tolerance genes will grow better than T7. This is when the salt concentration is 0.

If 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 at this time, GroESL is still faster than T7. But the control group is still the fastest because 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.

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 exceeded the control group and T7 with the highest growth efficiency. But T7 is still at the bottom. 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 verified 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 to improve salt tolerance, and C represents the control group without any insertion. From the following chart, it can be seen that the colonies formed by three different strains in saline gradually decrease with the increase of salt concentration.

The strain represented by Z has always formed the fewest colonies among the three strains, because it has not only inserted the gro promoter and the ire 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. When the concentration of saline reaches 20%, the strain represented by Z basically stops growing.

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. The strain represented by gro basically stopped growing when the saline concentration reached 25%.

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 gro.

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. When the concentration of bivalent cadmium is 10%, the percentage of bivalent cadmium absorbed is the largest among the strains represented by R-P. When the concentration of saline gradually increased, the rate of uptake of bivalent cadmium by strains represented by R-P also gradually decreased. When the concentration of bivalent cadmium reached 50%, the bivalent cadmium absorbed by the strains represented by R-P began to rise gently.

The percentage of bivalent cadmium absorbed by strains represented by R-E is always higher than that of strains represented by R-P. at 10, the percentage of bivalent cadmium absorbed by strains represented by R-P is the highest; At 25, the percentage of bivalent cadmium absorbed by R-P decreased a lot; When the concentration of bivalent cadmium increased from 25 to 50, the percentage of bivalent cadmium absorbed by R-P strain gradually increased. From 50 to 100, the percentage of bivalent cadmium absorbed by R-P strain finally gradually decreased.

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. This experiment verified that adding ire can greatly improve the ability of the strain to adsorb heavy metals.