Team:Jilin China/Parts
Parts
Our project includes five basic parts and three composite parts
| Name | Type | Description |
|---|---|---|
| BBa_K1932000 | Regulatory | The B.longum hup gene encodes the histone-like HU family protein HB1. |
| BBa_K1932001 | Translational_Unit | Orf and ori from Bifidobacterium |
| BBa_K1932002 | Protein_Domain | SEC2 signal peptides, putative serine protease, acid phosphatase domain |
| BBa_K1932003 | Protein_Domain | TMP1 signal peptides, hypothetical protein |
| BBa_K1932004 | Coding | TAT+apoptin |
| BBa_K1932005 | Device | This device is made up of BBa_K1932000;BBa_K1932001;BBa_K1932004 |
| BBa_K1932006 | Device | This device is constructed to express TAT-apoptin fused with sec2. |
| BBa_K1932007 | Device | This device is constructed for the expression of TAT-apoptin fused with tmp1. |
Parts
First, according to our design, the protein, Apoptin, should be expressed in Bifidobacterium. However, the promoters differ in various kinds of bacteria. BBa_K1932000, which contains the sequences of promoter and RBS of Bifidobacterium is included to regulate the expression of Apoptin. These sequences were originally cloned from the hup gene, which regulates the histone-like protein, HU, and they were proven to be able to regulate the expression of the exogenous genes in Bifidobacterium as well. We have registered this sequences as BBa_K1932000.
Parts
To replicate this recombinant plasmid in Bifidobacterium, the second part, BBa_K1932001, is included. This sequences was originally cloned from the plasmid in Bifidobacterium, which contains one ori and two orf. These two sequences ensures the replication and the stability of the recombinant plasmid in Bifidobacterium respectively.
Parts
The next part, BBa_K1932004, is essential for our project. It carries the sequence of TAT-Linker-Apoptin, which encodes the fusion protein, TAT-Apoptin. TAT is the transduction domain of HIV-1 virus which facilitates the associated protein to pass through the cell membrane. Apoptin is the protein which we use to treat solid tumors cells. Apoptin has been report to be able to induce the apoptosis of cancer cells and do no affect the growth of normal cells. Thereotically, this fusion protein is able to get into the cancer cells and induce the apoptosis.
Parts
In addition, we chose two signal peptides, Sec2 and Tmp1from B. breve UCC2003, to assist the secretion of the fusion protein from Bifidobacterium. The work of XX showed high activity of these two peptides in Bifidobacterium, and we confirmed the function of them with computer simulation of directing the behavior of secretion. We have registered them as BBa_K1932002 and BBa_K1932003.
Parts
With the aim of comparing the efficiency of the two signal peptides and the expression of our target protein attached to the signal peptides, we combined these three composite parts in different ways. BBa_K1932005 is the fusion protein only. BBA_1932006 and BBa_K1932007 are the fusion protein attached with SEC2 and TMP1 respectively. All these three parts are cloned with BBa_K1932000 and BBa_K1932001 to function as competent plasmids.
The three parts are cloned in the vector pgh and electrotransfered into Bifidobacterium. The expression of the fusion protein has been confirm with the western blot, and the results are shown as below.
