Difference between revisions of "Part:BBa K2922040"

Latest revision as of 21:09, 21 October 2019

Caveolin-1 coding region

Summary

This part contains the coding region of caveolin-1 (CAV1) gene, which naturally exists in many vertebrate cells. This gene could produce a kind of vesicle named caveolae in cell surfaces of mammals. According to a recent study, through heterologous expression of caveolin-1, heterologous caveolae (h-caveolae) will be observed in the cytoplasm of E. coli, where they can play the role of endocytosis (1,2). Therefore, we used caveolin-1 to perform endocytosis in E.coli.

Figure 1. Schematic diagram of action principle for CAV1

Biology and Usage

The CAV1 gene derived from the human genome was chosen. By codon optimization, the sequence suitable for expression in E. coli was constructed, and we hoped that it could exert endocytosis in E. coli. Furthermore, this part was used to construct several composite parts.

The coding cequence of target gene CAV1 was inserted into an expression vectors with T7 and RBS to obtain BBa_K2922042. We transformed the constructed plasmid into E. coli BL21 (DE3) to verify its successful heterologous expression.

Figure 2. The BBa_K2922042 was constructed to verify the expression in E.coli

Thereafter, BBa_K2922042 was connected with BBa_K346030 constructed by Peking University in 2010, and a new composite part BBa_K2922044 was obtained. In BBa_K346030, the construction of DsbA-MBP fusion protein can localize the metal-binding peptide for lead in the periplasm of E. coli. We may imagine, the cooperation of CAV1 and DsbA-MBP could improve the absorption ability of E. coli toward lead in the aqueous environment through enrichment.

Figure 3. The composite part BBa_K2922044 was constructed on the basis of DsbA-MBP and CAV1 to achieve higher absorption capacity of Pb(Ⅱ).

Identification

After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in Fig.4.

Fig 4.DNA gel electrophoresis of restriction digest products of Cav1-pUC57 (Xbal I & Pst I sites)

Characterization

1. Proof of the expression of CAV1

We used 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. 5). In this way, we successfully proved the heterologous expression of CAV1 in E.coli.

Fig. 5. (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.

2. Abilities of absorbing heavy metal ions

Since we wanted to use CAV1 to improve the absorption ability of E. coli toward lead, BBa_K2922044 was constructed by connecting BBa_K2922042 with BBa_K346030 (from 10_Peking). Therein, we hoped to combine the function of the fusion protein DsbA-MBP and CAV1, so that we could improve BBa_K346030.

Inductively coupled plasma mass spectrometry (ICP-MS) was used to accurately test the absorption amount of lead per unit mass of cells. BL21 (DE3) with DsbA-MBP and DsbA-MBP-CAV1 integration plasmids were separately cultured in medium containing 50 μM Pb(II). The amounts of lead absorbed by the bacteria with different circuits were measured. The relative absorption capacity of DsbA-MBP-CAV1 increased significantly and was much higher than that of DsbA-MBP. At the same time, the death and rupture of bacteria caused by the toxicity of Pb(II) may be the reason for absorption capacity decreased at 18 h in DsbA-MBP group. This demonstrated that the introduction of CAV1 could not only enhance the lead absorption efficiency, but also improve the resistance of bacteria against heavy metal. These data suggested us that CAV1 could play roles more than endocytosis.

Figure 6. Relative Pb（II）Absorption Capacity Per Unit Mass (DsbA-MBP & DsbA- MBP-Cav1)

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