Part:BBa_K3698008
RDLCK
rDegP+degP+Linker+amilCP+kan
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3238
Illegal BamHI site found at 3589 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2574
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2514
Illegal SapI site found at 2724
Experiment and Results
In order to facilitate screening during gene editing, we first need to add a kan resistance gene fragment containing FRT flanking downstream of the element degP_CP. First, pdegP_CP was linearized by PCR, and the linearized pdegP_CP 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 pKD13 plasmid as a template to amplify the kan resistance gene fragment containing FRT flanking by PCR. The 5'ends of the primers used respectively contain the homology arms with the recombination position of pdegP_CP. 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 pDCKF, the map is shown in Figure 4, containing a CmR resistance gene and fragments required for gene editing.
Figure 1. Linearized pdegP_CP gel electrophoresis image
Figure 2. kan_FRT gel electrophoresis image
Figure 3. Validation gel electrophoresis diagram of recombinant plasmid pDCKF
Figure 4. pDCKF plasmid map
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_FRT fragment was amplified from the pDCKF plasmid, inserted into the end of the stop codon of the degP gene of the MG1655 wild-type strain, and the kan resistance gene was knocked out to obtain the MG155_LCP strain. Amplify the degP_CP+kan_FRT (carrying homology arm) fragment from pDCKF plasmid and insert it into the 4639550bp position of MG1655_ΔdegP strain and name it MG1655_HDC strain; amplify the degP_CP+kan_FRT (carry homology arm) fragment from pDCKF plasmid and insert it into MG1655_ΔdegP strain The 2363163 bp position of MG was named MG1655_LDC strain.
Figure 5. Flow chart of gene editing strain construction Three strains of 3MG155_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 determine OD588 and OD600. After 12h, 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 degrees Celsius. At 45 degrees Celsius, 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. Dilute the bacterial solution cultivated to logarithmic phase at 37°C by 10, 100, and 1000 times and spread it on a non-resistant LB-agar medium. Each strain was spread on two groups, and one group was cultured at 37°C. The other group was incubated at 45°C. At about 18 hours, colony counts are performed. As shown in Figure 6, the number of colonies formed by the three strains cultured at 37°C was similar, while at 45°C, the number of colonies was MG1655_HDC>MG1655_LCP>MG1655_LDC. 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 6. Growth curves of the three strains at different temperatures
Figure 7. Plate image of colony count
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. At 45°C, the expression of degP was significantly different, 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℃
Figure9. The expression of degP of the three strains at 45℃
conclusion
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 plants.
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