Team:XHD-ShanDong-China/Description
Description
Escherichia coli, as an important model organism, plays an important role in current biological engineering. Since E. coli can produce human enzymes through recombinant DNA techniques, it is widely used to produce useful compounds or enzymes for medication (such as human insulin, vaccines etc.).
The application in modern industrial fermentation requires E. coli to have better tolerance, such as thermal adaptability. The optimal growth temperature for E. coli is 37°C, but in a fermentation factory, the operation of equipment and the metabolic process of E. coli will generate heat. Without intervention, the environmental temperature of E. coli will increase. When the temperature is higher than 37°C, there will be proteins in E. coli that fold errors or fail to fold normally, and excessive accumulation of these abnormal proteins will pose a fatal threat to E. coli. This will cause the loss of microbial fermentation industry.
Based on this, wo hope to improve the thermal adaptability of Escherichia coli through synthetic biological means.
In the periplasmic space of Escherichia coli, the CpxR protein will be phosphorylated in a high temperature environment, and the phosphorylated CpxR will bind to the upstream of the degP gene and regulate the transcription of degP together with the σ24 transcription factor which is expressed by the rpoE gene. The DegP protein expressed by degP is an essential protein for the high temperature growth of E. coli, also known as HtrA (High temperature requirement A). It is responsible for the digestion of abnormally folded proteins in the periplasmic space in a high temperature environment, which is essential for the thermal adaptability of E. coli. The regulatory relationship between cpxR, rpoE and degP belongs to the FFL(feedforward loop) network motif, as show in Figure 1.
Figure 1. Schematic diagram of FeedForward Loop motif. The Expression of degP is coregulated by CpxR and rpoE.
Our project aims to modify the wild-type E. coli so that it can maintain activity even at higher temperature. Traditional synthetic biology methods such as enhanced promoter, plasmid transfection and other methods have problems such as waste of cell resources and loss of plasmids. We hope to find a more stable method to improve the temperature adaptability of E. coli. After consulting the literature and experts in related fields, we learned that the expression regulation between genes in E. coli is affected by the spatial distance between genes. Therefore, we hope to regulate the expression of heat shock genes by adjusting the distance between heat shock genes and their regulatory genes, thereby regulating the thermal adaptability of E. coli. At the same time, exploring the relationship between the distance and expression of genes in the network motif and provides a new research idea for synthetic biology.
Based on the chromosome three-dimensional structure model of E. coli, we determined the spatial distances between degP gene and cpxR or rpoE. On this basis, we changed the position of the degP gene through λ-Red technology to change the spatial distances between degP and cpxR or rpoE genes. We fused a blue chromoprotein to degP. By detecting the absorbance of the blue chromoprotein, we can get the expression level of degP, and we can get the growth curve of E. coli by the absorbance of 600nm. Comparing the expression levels of degP and growth curves of different strains in normal and high temperature environments, we can know the influence of the distance between genes in the motif on gene expression, and the influence of degP expression on the thermal adaptability of E. coli. In the end, we can filter to get E. coli strain with better thermal adaptability.
We established a mathematical model to simulate the expression regulation relationship between genes in the network motif at different gene distances, which provided theoretical support for the overall design of the experiment. At the same time, fitting the model with experimental data also further verified the correctness of the model.
Bacteria are often used in scale-up production. In the process of fermentation, the temperature rises easily above the optimal temperature due to the intense metabolite activities of cell. So we need to use a lot of water to cool the fermentation tank or the microbes would die or stop working. However, if we use this genetically-edited bacteria which is able to survive a higher temperature without impairing the productivity, the cost of cooling will be reduced considerably. In addition, the improvement will not be lost over generations because the genome of the bacteria is fundamentally edited which is very convenient in practical use.
In addition, Some iGEM teams intended to tackle environmental issues using bacteria. But some places with high temperature, like Ethiopia in Africa, are not an ideal environment for their microbiologic tools. In that case, our design to increase the heat adaptability of bacteria by changing distances between genes might provide a innovative solution.
