View source for Part:BBa K2922042


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<partinfo>BBa_K2922042 short</partinfo>

===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''.
<table><tr><th>[[Image:Im-1.png|thumb|450px|Fig 1. Schematic diagram of action principle for CAV1]]</th><th></table>

===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).
<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>


===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).
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2.	J. Shin et al., Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids. ACS Synthetic Biology 8, 1055-1066 (2019).


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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2922042 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K2922042 parameters</partinfo>
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