Difference between revisions of "Part:BBa K2922040"

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<table><tr><th>[[Image:Im-1.png|thumb|450px|Figure 1. Schematic diagram of action principle for CAV1]]</th><th></table>
 
<table><tr><th>[[Image:Im-1.png|thumb|450px|Figure 1. Schematic diagram of action principle for CAV1]]</th><th></table>
 
It has been shown in the literatures (1,2) that the formation of heterologous caveolae (h-caveolae) can be observed in the cytoplasm of ''E. coli'' after the heterologous expression of caveolin-1. Thus, we chose CAV1 to perform endocytosis in ''E.coli''.
 
It has been shown in the literatures (1,2) that the formation of heterologous caveolae (h-caveolae) can be observed in the cytoplasm of ''E. coli'' after the heterologous expression of caveolin-1. Thus, we chose CAV1 to perform endocytosis in ''E.coli''.
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===Identification===
 
===Identification===
 
After receiving the synthesized Part DNA, restriction digest identification was done to certify the plasmid is correct, and and the experimental results were shown in Fig. 1. A separate fragment is 537 bp.
 
After receiving the synthesized Part DNA, restriction digest identification was done to certify the plasmid is correct, and and the experimental results were shown in Fig. 1. A separate fragment is 537 bp.
 
<table><tr><th>[[Image:Im-2.png|thumb|150px|Fig 2.DNA gel electrophoresis of restriction digest products of DH-Cav1-pUC57 (Xbal I & Pst I sites)]]</th><th></table>
 
<table><tr><th>[[Image:Im-2.png|thumb|150px|Fig 2.DNA gel electrophoresis of restriction digest products of DH-Cav1-pUC57 (Xbal I & Pst I sites)]]</th><th></table>
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===Verify the expression of CAV1===
 +
We usd T7 promoter to highly express CAV1 in ''E. coli'' in our composite part BBa_K2922042. After heterologous protein expression, no target bands were observed through SDS-PAGE. However, according to literature, vesicles formed from CAV1 on the inner membrane of ''E. coli'' which could hardly be detected through SDS-PAGE technique. So, 5(6)-carboxyfluorescein, a fluorescent molecule, which could pass through the outer membrane but not the inner membrane (2) was selected to check if it can perform endocytosis. After induction, 5(6)-carboxyfluorescein was added to the medium and cultured for 24 h. As shown in Fig. 1, compared with the faint yellow color in control group, significant color change (orange-yellow color) was observed by naked eyes in the CAV1 group, which came from fluorescent dye. The rod-shaped fluorescence appeared in the CAV1 group obviously, and its relative position was consistent with that of bacteria in the bright field (Fig. 2).
 +
<table><tr><th>[[Image:Im-3.png|thumb|700px|Fig. 2. (A) Different colors of experimental group (BBa_K2922042) and control group (BBa_K525998) were shown after incubation with 5(6)-carboxyfluorescein, regardless of the operations of centrifugation or resuspension. (B) The photos taken by fluorescence microscopy showed significant difference between experimental group and control group. ]]</th><th></table>
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===Reference===
 
===Reference===

Revision as of 14:51, 21 October 2019


Caveolin-1 coding region

Summary

This part contains the coding region of caveolin-1 gene. Caveolae are endocytic pits located in mammalian cell surfaces where they play indispensible roles in endocytosis.

Figure 1. Schematic diagram of action principle for CAV1

It has been shown in the literatures (1,2) that the formation of heterologous caveolae (h-caveolae) can be observed in the cytoplasm of E. coli after the heterologous expression of caveolin-1. Thus, we chose CAV1 to perform endocytosis in E.coli.


Identification

After receiving the synthesized Part DNA, restriction digest identification was done to certify the plasmid is correct, and and the experimental results were shown in Fig. 1. A separate fragment is 537 bp.

Fig 2.DNA gel electrophoresis of restriction digest products of DH-Cav1-pUC57 (Xbal I & Pst I sites)


Verify the expression of CAV1

We usd T7 promoter to highly express CAV1 in E. coli in our composite part BBa_K2922042. After heterologous protein expression, no target bands were observed through SDS-PAGE. However, according to literature, vesicles formed from CAV1 on the inner membrane of E. coli which could hardly be detected through SDS-PAGE technique. So, 5(6)-carboxyfluorescein, a fluorescent molecule, which could pass through the outer membrane but not the inner membrane (2) was selected to check if it can perform endocytosis. After induction, 5(6)-carboxyfluorescein was added to the medium and cultured for 24 h. As shown in Fig. 1, compared with the faint yellow color in control group, significant color change (orange-yellow color) was observed by naked eyes in the CAV1 group, which came from fluorescent dye. The rod-shaped fluorescence appeared in the CAV1 group obviously, and its relative position was consistent with that of bacteria in the bright field (Fig. 2).

Fig. 2. (A) Different colors of experimental group (BBa_K2922042) and control group (BBa_K525998) were shown after incubation with 5(6)-carboxyfluorescein, regardless of the operations of centrifugation or resuspension. (B) The photos taken by fluorescence microscopy showed significant difference between experimental group and control group.


Reference

1. J. Shin et al., Display of membrane proteins on the heterologous caveolae carved by caveolin-1 in the Escherichia coli cytoplasm. Enzyme Microb Technol 79-80, 55-62 (2015).
2. J. Shin et al., Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids. ACS Synthetic Biology 8, 1055-1066 (2019).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]