Revision as of 20:00, 19 October 2016

This device is constructed to express TAT-apoptin fused with sec2.

This device was cloned to express the fusion protein, TAT-Apoptin and the Sec2 peptide coding sequence was included to increase the secretion of the protein. Among the subparts, BBa_K1932000 is a strong promoter to regulate the expression of exogenous protein in Bifidobacterium. BBa_K1932001 is included to enhance the stability of the device in Bifidobacterium. BBa_K1932002 is the sequence of the signal peptide, and Sec2 can direct the export of the protein from Bifidobacterium. BBa_K1932004 encodes the TAT-apoptin fusion protein, which can induce the apoptosis specifically in the cancer cells.

Characterization:

We have simulated the structure of the fused protein. The signal peptide prediction was performed by SingalP 4.1 Server (Fig.1), TMpred program (Fig.2) and TMHMM (Fig.3), and the results showed that it could direct the process of secretion by cutting the site between amino acid 34 and 35.

The three-dimensional structure of our protein was simulated by homology modeling and molecular dynamic simulation using the Phyre2 web portal for protein modeling and Hyperchem 8.0 (Fig.4), which showed that the two domains were separate, indicating that the function of both TAT-Apoptin and sec2 would not be affected by each other.

The part of BBa_K1932006 was synthesized and cloned in a pGH vector by Generay Biotechnology. The plasmid was cut by the restriction enzymes, EcoRⅠ and PstⅠ, and separated by 1% agarose gel(Fig.5).

The sequence was ligated into the vector pSB1C3 by T4 ligase at 16℃ overnight,and the ligated construct was transformed into the E.coli(Fig.6).

To ensure the insertion of the right-size sequence, the sequence was cut again and tested by agarose gel electrophoresis (Fig.7).

Once the size of this sequence was confirmed, the bacteria containing the construct were sent to the Comate Bioscience Company for DNA sequencing for further verification. The detailed protocols of these experiments were shown in table 1 and table 2.

The device was transformed into competence bacterium DH5α for amplification, and extracted and purified with the Plasmid Minipreparation Kit from BioTeke. To test the usage of our device in different environment, the expression of the protein, examined with the method of SDS-PAGE, in E.coli was assisted by BIT-China (Fig.7).

Furthermore, the device was transformed into the Bifidobacterium longum with electrotransformation (1800v,15μf,200Ω), and the transformed Bifidobacterium were incubated in anaerobic incubator for approximately 24 hours. According to the results of cell experiments, we decided to use the Bifidibacterium with this device in the form of lyophilized powders and they were used to test the function of our device in vivo.

After constructing the cancer model by injecting SMMC-7721 cell suspension, the mice were divided into five groups. One of these group was treated with the Bifidobacterium longum that transformed with this device (injection in situ). After 21 days, the mice were killed by cervical dislocation and the tumor mass was measured and weighed (Fig.8 and Fig.9).

Sequence and Features