Team:Tel-Hai/Proof

iGEM Tel-Hai 2016

Demonstrating A Proof of Concept

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

    5. * 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.