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From 2013.igem.org

Design and assembly: We aim to ensure that the specifications drawn are considered in the design.

In recent studies, Apoptin has been described to induce apoptosis in various human cancer cell lines. To develop apoptin as an anti-cancer drug, efficient transduction tools have been used to deliver apoptin into tumor cells. cell-penetrating peptides, such as the trans-acting activator of transcription (TAT) protein transduction domain (PTD) and MPG peptide have therefore been used as vectors for protein delivery.So we decided apoptin fused to TAT peptide because this peptide move apoptin into cell without membrane damage. We fused Hemoglutinin A with TAT in order to decrease possibility of getting trapped in microcytosis.In addition to this, we conceived an alternative technology that offers fusing another cell penetrating peptide with apoptin, thus we constructed MPG peptide with apoptin and we applied that idea again, on another protein which is named E4of4. E4of4 is known to induce apoptosis as it is a tumor specific molecule. We demonstrated E4of4 fused to TAT peptide as a tumor specific penetrating molecule .As a result, we brought MPG-Apoptin and TAT-E4of4 into the literature.

To apply the project, we ordered apoptin fused to TAT, TAT HA and MPG sequences respectively as a composite part in order to minimalize cost and time. We cloned the genes into pSB1C3 backbone. After cloning, we expressed the proteins in BL21. coli strain under constutive promoter firstly.İn addition to this,our constructs were harboring a 6xHis tag. The 6×His were fused with constructs at the N-terminus to enhance protein purification.

After cultivatization, we controlled our genes in gel electrophoresis. Electrophoresis proved the transformation of new genes was successful. There are gel electrophoresis results especially content of Module 2 below:

Fotogtraf!!!!!!!!