Latest revision as of 04:20, 21 October 2016

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

This device was improved from the part of BBa_K1166005(https://parts.igem.org/Part:BBa_K1166005) registered by TecMonterrey(http://2013.igem.org/Team:TecMonterrey) and 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:

This device was improved from the part of BBa_K1166005(https://parts.igem.org/Part:BBa_K1166005) registered by TecMonterrey(http://2013.igem.org/Team:TecMonterrey), which encodes the fusion protein, TAT-apoptin. To use the biobrick in Bifidobacterium, BBa_K1932000,BBa_K1932001 and BBa_K1932002 were added. Compared with the old part, the device could stably exist and replicate in Bifidobacterium and the expression of TAT-apoptin in Bifidobacterium was up-regulated. In addition, the TAT-apoptin could be secreted out of the Bifidibacterium with the help of the signal peptide, Sec2.

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.

Fig.1. Analysis of signal peptide on Sec2 by the SignalP 4.1 Server

Fig.2. Analysis of transmembrane-spinning region on Sec2 by the TMpred

Fig.3. Analysis of hydrophobic region on Sec2 by the TMHMM

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.

Fig.4. The simulated structures for Sec2-TAT-Linker-apoptin (Sec2 are highlighted in yellow).

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).

Fig.5. (1) Marker; (2) pGH+Sec2 digested with EcoRⅠ and PstⅠ

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).

Fig.6. (1) control(only DH5α);(2)DH5α transformed with BBa_K1932006 (the Sec2-device+pSB1C3 vector)

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

Fig.7.Different temperature induced with precipitation

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 to Fig.10).

Fig.8.The volume of the tumor was measured and recorded every three days. Negative control group were injected 1X PBS buffer. Positive control group were injected doxorubicin hydrochloride at a dosage of 4.3769mg/kg. BF group were injected B. Longum at a dosage of 2.5×10Λ7 CFU/k. BFS group were injected B. Longum with BBa_K1932006 at a dosage of 2.5×10Λ7 CFU/kg.

Fig.9.The mice were killed by cervical dislocation in 21d and the tumors were weighed. Positive control group were injected doxorubicin hydrochloride at a dosage of 4.3769mg/kg. BF group were injected B. longumat adosage of 2.5×10Λ7 CFU/k.

Fig.10.The mice were killed by cervical dislocation in 21d and the tumors were weighed. Positive control group were injected doxorubicin hydrochloride at a dosage of 4.3769mg/kg. BF group were injected B. Longum at a dosage of 2.5×10Λ7 CFU/k.

Sequence and Features