Difference between revisions of "Part:BBa K2922042"

(→‎Verify the expression of CAV1)
 
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===Summary===
 
===Summary===
−
This part is constructed to verify the expression of caveolin-1. It could form vesicles in the inner membrane and perform endocytosis in ''E.coli''.
+
This part was constructed to verify the expression of caveolin-1 in ''E.coli'', in the inner membrane of which it could form vesicles and perform endocytosis.
 
<table><tr><th>[[Image:Im-1.png|thumb|450px|Fig 1. Schematic diagram of action principle for CAV1]]</th><th></table>
 
<table><tr><th>[[Image:Im-1.png|thumb|450px|Fig 1. Schematic diagram of action principle for CAV1]]</th><th></table>
  
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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.
 
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).
 
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>
+
<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.Scale bar = 1 μm. ]]</th><th></table>
  
 
===Conclusion===
 
===Conclusion===
−
Our experiment demonstrated that the introduction of CAV1 performed endocytosis in E. coli successfully.
+
Our experiment demonstrated that the introduction of CAV1 performed endocytosis in ''E. coli'' successfully.
  
  

Latest revision as of 14:54, 21 October 2019


The high level expression of BBa_K2922040 by T7 Promoter

Summary

This part was constructed to verify the expression of caveolin-1 in E.coli, in the inner membrane of which it could form vesicles and perform endocytosis.

Fig 1. Schematic diagram of action principle for CAV1

Verify the expression of CAV1

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.Scale bar = 1 μm.

Conclusion

Our experiment demonstrated that the introduction of CAV1 performed endocytosis in E. coli successfully.


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]