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Team:Kyoto/CiC/Method
From 2009.igem.org
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==Method== | ==Method== | ||
| - | ===Subgoal 1=== | + | ===Subgoal 1 Liposomes with HIV-1 TAT peptide=== |
Subgoal A is construction of liposome that penetrate through human cell membranes. | Subgoal A is construction of liposome that penetrate through human cell membranes. | ||
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To acheive this, we designed ''' liposomes with HIV-1 TAT peptide.''' | To acheive this, we designed ''' liposomes with HIV-1 TAT peptide.''' | ||
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HIV-1 TAT is a Protein Transduction Domain(PTD). | HIV-1 TAT is a Protein Transduction Domain(PTD). | ||
Liposomes made of peptidolipid with HIVｰ1 TAT is known to penetrate through cell membrane. | Liposomes made of peptidolipid with HIVｰ1 TAT is known to penetrate through cell membrane. | ||
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And the TAT side of the protein will be exposed to water so that TAT domain can touches the cell membrane. | And the TAT side of the protein will be exposed to water so that TAT domain can touches the cell membrane. | ||
When exposed to cells, liposomes with this fusion protein will promote endocytosis and be taken into the cell. | When exposed to cells, liposomes with this fusion protein will promote endocytosis and be taken into the cell. | ||
| + | If such liposomes are fluorescent stained, they will show fluorescence in the cells. | ||
We constructed plasmids encoding this fusion protein. | We constructed plasmids encoding this fusion protein. | ||
When this plasmid is mixed with mixture of liposomes and cell-free protein synthesis system, the fusion proteins will be synthesized and anchored on Liposomes. | When this plasmid is mixed with mixture of liposomes and cell-free protein synthesis system, the fusion proteins will be synthesized and anchored on Liposomes. | ||
| - | + | We also performed similar experiments with chemically synthesized TAT-LALAAAA protein. | |
| - | + | ||
===Subgoal 2=== | ===Subgoal 2=== | ||
Revision as of 19:41, 20 October 2009
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- Mechanism
Method
Subgoal 1 Liposomes with HIV-1 TAT peptide
Subgoal A is construction of liposome that penetrate through human cell membranes. To acheive this, we designed liposomes with HIV-1 TAT peptide.
HIV-1 TAT is a Protein Transduction Domain(PTD). Liposomes made of peptidolipid with HIVｰ1 TAT is known to penetrate through cell membrane. It is suggested that HIV-1 TAT promotes endocytosis,though the precise mechanism remains unclear.
Then we fused HIV-1 TAT with potential anchor protein (LALAAAALALAAAALALAAAA). The LALAAAA side of this fusion protein is expected to be anchored in the lipid barrier of liposome. And the TAT side of the protein will be exposed to water so that TAT domain can touches the cell membrane. When exposed to cells, liposomes with this fusion protein will promote endocytosis and be taken into the cell. If such liposomes are fluorescent stained, they will show fluorescence in the cells.
We constructed plasmids encoding this fusion protein. When this plasmid is mixed with mixture of liposomes and cell-free protein synthesis system, the fusion proteins will be synthesized and anchored on Liposomes.
We also performed similar experiments with chemically synthesized TAT-LALAAAA protein.
Subgoal 2
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Subgoal 3
this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.this is the subgoal.
