Team:XHD-ShanDong-China/Design
Design
Our project aims to change the spatial distances between the degP gene and the other two genes in the feedforward loop by gene editing, explore the influence of spatial distance on the dynamics of the network motif, and try to screen out strain with better thermal adaptability. As shown in the figure 1, in the three-dimensional structure model of E. coli chromosome, the distance between the original position of degP and cpxR is 7.3. When degP move to Z1 position, the distance to cpxR is 6.3. When degP move to Z2 position, the distance to cpxR is 11.0. Theoretically, degP-deficient strains have very low activity at high temperature; when degP moves to the Z1 position, the distance to cpxR becomes closer, and CpxR can start the transcription of degP more quickly when E. coli at high temperature. Thus the strain can adapt to the high temperature environment.We have established a mathematical model to simulate the expression regulation relationship between genes in the network motif, and by calculating the highest concentration of the protein expressed by the degP gene under different spatial distances to cpxR, to explore the relationship between the spatial distance between genes and gene expression.
Figure 1. Schematic diagram of 3D model of changing gene distance.
We need to change the location of the degP gene by gene editing. First we need to construct a composite plasmid to prepare the fragment for insertion into the MG1655 genome. As shown in Figure 2, the composite plasmid pRDLCK contains the degP regulatory region rDegP, a degP fused with amilCP by Linker, and a kan resistance genes.
Figure 2. Plasmid map of pDCKF.
In this project, 4 strains were obtained through gene editing, as shown in Figure 3. The regulatory region rDegP and degP genes of the degP gene were amplified by PCR from wild-type E. coli MG1655 and fused with the chromoprotein amilCP to form RdegPLCP. The kan resistance gene amplified by PCR from pKD13 was inserted into the downstream of RdegPLCP to constitute the RDLCK. The Linker+amilCP+kan fragment named as LCP_kan was amplified from RDLCK and inserted into the position before the degP stop codon of the MG1655 genome to form the MG1655_LCP strain. This strain did not change the position of the degP gene, and only fused the chromoprotein amilCP to degP. Before moving the position of degP, we first knocked it out from the genome of the wild-type strain MG1655 by the λ-Red to obtain the degP-deficient strain MG1655_ΔdegP. Amplify the rDegP+degP+Linker+amilCP+kan fragment named as LDCK and HDCK with different homology arms from RDLCK, insert HDCK into the Z1 position of the MG1655 genome, as show in Figure 1, and obtain the MG1655_HDC strain. The spatial distance between degP and cpxR in this strain is reduced. In theory, it has high thermal adaptability. Insert LDCK into the Z2 position of the MG1655 genome to obtain the MG1655_LDC strain. The spatial distance between degP and cpxR in this strain is enlarged. Theoretically, the thermal adaptability will be lower than that of the wild type.
Figure 3. Schematic diagram of experimental design flow chart.
