Latest revision as of 08:38, 23 October 2016

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

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) was 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.

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_K1932003 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, Tmp1.

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.

Fig.1. Analysis of signal peptide on Tmp1 by the SignalP 4.1 Server

Fig.2. Analysis of transmembrane-spinning region on Tmp1 by the TMpred

Fig.3. Analysis of hydrophobic region on Tmp1 by the TMHMM

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.

Fig.4. The simulated structures for Tmp1-TAT-Linker-apoptin (Tmp1 are highlighted in yellow.)

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).

Fig.5. (1): Marker; (2) pGH+TMP1 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_K1932007 (the TMP1-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. (1) Marker; (2) BBa_K1932007 (pSB1C3+TMP1-device) digested with EcoRⅠ and PstⅠ

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. After the results coming out, the sequence turns out to be correct.

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).

Fig.8 Different temperature induced with precipitation

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,Fig.10 and Fig.11).

Fig.9.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. BFT group were injected B. Longum with BBa_K1932007 at a dosage of 2.5×10Λ7 CFU/kg.

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. BFT group were injected B. Longum with BBa_K1932007 at a dosage of 2.5×10Λ7 CFU/k.

Fig.11.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. BFT group were injected B. Longum with BBa_K1932007 at a dosage of 2.5×10Λ7 CFU/k.

Sequence and Features