Part:BBa_K1166005:Experience

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Applications of BBa_K1166005

Team: 2016 Jilin_China team

Improvment: We were inspired by the pioneer work from the TecMonterrey team of 2013 iGEM competition(http://2013.igem.org/Team:TecMonterrey) , which demonstrated that the TAT-Apoptin could kill tumor cells. The parts, BBa_K1932005 (https://parts.igem.org/Part:BBa_K1932005), BBa_K1932006 (https://parts.igem.org/Part:BBa_K1932006) and BBa_K1932007 (https://parts.igem.org/Part:BBa_K1932007) were constructed based on BBa_K1166005(https://parts.igem.org/Part:BBa_K1166005).

Although TAT-Apoptin could induce the apoptosis of tumor cells effectively, it seemed that E. coli was not asuitablevehicle to deliver TAT-Apoptin into tumors. We thought thatanaerobic microorganisms might be more effective than E. coli totarget the tumor tissues. After extensive literature searching and consulting with experts from differentresearch fields, we found that Bifidobacterium longum (B.longum) should be an ideal vehicle to deliver TAT-Apoptin. First, B. longum only grows in anaerobic regions, which means that the expression of TAT-Apoptinfrom B. longumcan only happen in anaerobic regions, such as the anaerobic regions inside solid tumors. More importantly,B.longumissafe to human body since it produces no toxin to the host. Furthermore, it has anti-tumor biological effects. Considering all these information, we have chosenB. longum instead of E.coli to express the TAT-Apoptin.

To facilitate the expression of TAT-apoptin in B. longum, many biobricks have been replaced or added in the devices. First of all, Tmp1, originally cloned from the plasmid of Bifidobacterium that contains one ori and two orf sequences was added as the first biobrick to regulate the expression of the TAT-apoptin in B.longum.In addition, the promoter and RBS sequence from hup gene of B.longum that could function in the recombinant plasmid inside B. longumwas also added to regulate the expression of this protein. To facilitate the secretion of TAT-apoptin, the signal peptides of Sec2 and Tmp1 were added to direct the secretion process of the fused protein in two separate devices. To confirm the function of TAT-Apoptin and extend the work of the TecMonterrey team, the two devices were transformed into B.longum and the animal experiments were done, which showed that our idea is feasible. More details about the experiments are available in our wiki.

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