Team:XHD-ShanDong-China/Proof Of Concept
Proof of Concept
In order to prove that the distances from the Z gene to the X and Y genes in the feedforward loop network motif will affect the expression of Z gene, we established a mathematical model to simulate the expression of X, Y and Z proteins under different gene spatial distances. The regression curves of gene spatial distance and Z-protein expression concentration were established to predict the amount of Z-protein expression under different gene spatial distances and to infer the change of thermal adaptation of E. coli under different gene distances, thus providing the theoretical basis and design direction for the overall experimental design. We used the network motif of cpxR, rpoE, and degP as an example to change the position of the Z gene degP by means of gene editing, thereby changing the distances between degP and the other two genes. After the change in distance, the expression of degP will also change accordingly. Since degP is an important heat-adaptation-related gene, we also hoped to screen relatively well-adapted strains for use in the microbial fermentation industry by varying the distance between degP and its regulatory genes.
In order to facilitate screening during gene editing, we first need to add a kan resistance gene fragment at downstream of the RdegPLCP. First, pRdegPLCP was linearized by PCR, and the linearized pRdegPLCP fragment was 4583 bp. As shown in Figure 1, the gel electrophoresis identification result met expectations, and the gel was cut and recovered. Then use the plasmid pKD13 as a template to amplify the kan resistance gene fragment by PCR. The 5' ends of the primers used in PCR respectively contain the homology arms with the recombination position of pRdegPLCP. The target fragment is 1364 bp, as shown in Figure 2. The gel electrophoresis identification result met expectations, and the gel was cut and recovered. The above two fragments were homologously recombined to generate a new plasmid. PCR verified that the target fragment was 1120bp. As shown in Figure 3, the gel electrophoresis result was in line with expectations, confirming the success of the recombinant plasmid. The recombinant plasmid was named pRDLCK, the map is shown in Figure 4, containing a CmR resistance gene and fragments required for gene editing.
Figure 1. Gel electrophoresis of linearized pRdegPLCP.
Figure 2. Gel electrophoresis of kan fragment.
Figure 3. Gel electrophoresis of verification of recombinant plasmid pRDLCK.
Figure 4. Plasmid map of pRDLCK.
In order to verify the relationship between distance and expression, we need 3 editing strains and select the strains with the best thermal adaptability from the 3 strains. The strain construction process is shown in Figure 5. The Linker+amilCP+kan fragment named as LCP_kan was amplified from the pDCKF plasmid, inserted into the position before the stop codon of the degP gene of the MG1655 wild-type strain to form the MG155_LCP strain. 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 Design-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 5. Flow chart of gene editing strain construction.
Three strains of MG155_LCP, MG1655_HDC, and MG1655_LDC were respectively inoculated into non-anti-LB liquid medium. Each strain was cultured in 3 tubes in a 37℃ incubator and 3 tubes in a 45℃ incubator. Samples were taken every 1h to measure OD588 and OD600. After 11h, the growth curves of the three strains were obtained. As shown in Figure 6, the curves of the three strains are not much different at 37°C. At 45°C, the three curves show more obvious differences. The growth curve of MG1655_HDC is the highest, MG1655_LCP in the middle, MG1655_LDC is the lowest.
Figure 6. Growth curves of the three strains at different temperatures.
Dilute the bacterial solution cultivated to logarithmic phase at 37°C by 10, 100, and 1000 times and spot 100uL on a non-resistant LB-agar plate. Each strain was spread on two groups, and one group was incubated at 37°C. The other group was incubated at 45°C. After about 18 hours, the colonies formed by live bacteria are shown in Figure 7. At 37°C, the numbers of viable bacteria of the three strains are relatively large, and the number of colonies grown is not much different. At 45°C, the number of viable bacteria showed a relatively obvious gradual trend. MG1655_HDC (degP closest to cpxR) had the largest number of live bacteria, and MG1655_LDC (degP closest to cpxR) had the least number of live bacteria. It can be seen that after our transformation, MG1655_HDC has the highest thermal adaptability, and this strain can be applied to microbial fermentation factory.
Figure 7. Live bacteria count chart.
OD588/OD600 was used to characterize the expression of degP. As shown in Figure 8, when cultured at 37°C, the expression of degP of the three strains was similar. As shown in Figure 9, the expression of degP at 45°C was significantly different, the degP expression of all three strains were higher than 37°C, and the expression of degP in three strains showed a trend of MG1655_HDC > MG1655_LCP > MG1655_LDC. It can be seen that changing the distance between the Z gene and the XY gene in the feedforward loop motif can change its expression, which provides a new idea for future synthetic biology research.
Figure 8. The expression of degP of the three strains at 37℃.
Figure 9. The expression of degP of the three strains at 45℃.
The results confirm that in the feed-forward loop network motif, when the spatial distance between the Z gene and its regulatory genes becomes longer, its expression will become weaker, and when the spatial distance becomes closer, its expression will increase. When the expression of degP is increased, the thermal adaptability of E. coli will be higher, and when the expression of degP is weakened, the thermal adaptability of E. coli will be weakened. The strains that we change the gene position can be used as engineered strains in microbial fermentation factory.
