Difference between revisions of "Part:BBa K3698008"

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<partinfo>BBa_K3698008 parameters</partinfo>
 
<partinfo>BBa_K3698008 parameters</partinfo>
 
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<h2> Experiment and Results </h2>
 
<h2> Experiment and Results </h2>
  
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.
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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.
  
 
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Figure 1. Linearized pdegP_CP gel electrophoresis image
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Figure 1. Gel electrophoresis of linearized pRdegPLCP.
  
 
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Figure 2. kan_FRT gel electrophoresis image
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Figure 2. Gel electrophoresis of kan fragment.
  
 
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Figure 3. Validation gel electrophoresis diagram of recombinant plasmid pDCKF
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Figure 3. Gel electrophoresis of verification of recombinant plasmid pRDLCK.
  
 
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   <img src="https://2020.igem.org/wiki/images/2/21/T--XHD-ShanDong-China--Proof-Figure4.jpeg" style="width:60%">
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   <img src="https://2020.igem.org/wiki/images/0/03/T--XHD-ShanDong-China--Design-Figure2.jpeg" style="width:60%">
  
 
   
 
   
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Figure 4. pDCKF plasmid map
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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_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.
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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.
  
 
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Figure 5. Flow chart of gene editing strain construction
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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.
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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.
  
 
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Figure 6. Growth curves of the three strains at different temperatures
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Figure 6. Growth curves of the three strains at different temperatures.
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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.
  
 
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Figure 7. Plate image of colony count
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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. 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.
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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.
  
 
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   <img src="https://2020.igem.org/wiki/images/4/40/T--XHD-ShanDong-China--Proof-Figure9.jpeg" style="width:60%">
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Figure 8. The expression of degP of the three strains at 37℃
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Figure 8. The expression of degP of the three strains at 37℃.
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Figure9. The expression of degP of the three strains at 45℃
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Figure 9. The expression of degP of the three strains at 45℃.
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<h2>conclusion</h2>
 
<h2>conclusion</h2>
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.
+
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.

Latest revision as of 02:30, 28 October 2020


RDLCK

rDegP+degP+Linker+amilCP+kan

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3238
    Illegal BamHI site found at 3589
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2574
  • 1000
    INCOMPATIBLE 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 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℃.


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 factory.