Difference between revisions of "Part:BBa K1932007"

Revision as of 18:14, 19 October 2016

This device is constructed for the expression of TAT-apoptin fused with tmp1.

This device is constructed for the expression of the fusion protein, TAT-Apoptin, and Tmp1 is included to increase the secretion of the protein. Among the subparts, BBa_K1932000 is added as a promoter to regulate the expression of exogenous protein in Bifidobacterium. BBa_K1932001 is included into the device to increase the stability of the device in Bifidobacterium. BBa_K1932003 encodes the signal peptide, Tmp1, which can direct the export of the protein from Bididobacterium. BBa_K1932004 encodes TAT-Apoptin, which acts as the effector protein to kill the cancer cells.

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 26 and 27.

The three-dimensional structure of our protein was simulated by homology modeling a 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 Tmp1 would not be affected by each other.

The part of BBa_K1932007 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 amplified in the competence bacterium DH5α, and was extracted and purified with the Plasmid Minipreparation Kit from BioTeke. To test the usage of our device in different laboratories,the expression of the protein was examined by the BIT-China with the method of SDS-PAGE (Fig.8).

After establishing the cancer model by injecting SMMC-7721 cell suspension into nude mice, the mice were divided into five groups. One of these group was treated with the Bifidobacterium longum transformed with this device. After 21 days, the mice were killed by cervical dislocation and the tumor mass was weighed and measured (Fig.9 and Fig.10).

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