Difference between revisions of "Team:Tel-Hai/Proof"

In order to explain our proof of concept, it's necessary to remind ourselves the main project goal: "Development of a novel and efficient delivery-system for gene editing technology in Cystic Fibrosis"

Work Plan:

1. BBa_K2061005 consist: B-subunit of the E. coli Heat-Labile Toxin (LTB), linker and short viral DNA binding domain (DBD) sequences, expression via pSB1C3 plasmid in E. coli.
2. Isolation of the new chimeric protein: LTB + DBD (LTBD).
3. LTBD binding to GFP plasmid.
4. LTBD + plasmid enter the cell when introduced to NCI-H1650 (human lung epithelial cells expressing ganglioside receptors on their plasma membrane).

* LTBD complex was pre incubated with and plasmid tagged with Hoechst for identification.

Our Proof of Concept Goals:

LTBD binding, delivery and expression of GFP plasmid to NCI-H1650:

1. LTBD bind GFP plasmid DNA
2. LTBD bind to ganglioside receptor
3. LTBD + GFP plasmid enter the cell via endocytosis
4. Plasmid go to the nucleus
5. GFP gene expression

Results:

1. LTBD stop DNA migration by binding to the plasmid.
   
2. LTBD with marked with green FITC attaching to cell membrane
   
3. LTBD in cell's cytosol
4. DNA in the nucleus
   

* We were unable to achieve the 5th goal due lack of time.

We want to thank Dr. Moshik Kutner-Cohen and Dr. Niv Bachnoff for guiding us to achieve all above.