Spot On Treatment Team Stockholm iGEM 2010 Andreas Constantinou Hassan Foroughi Asl Emmelie Lidh Johan Nordholm Nina Schiller söndag den 7 november 2010
Vitiligo ‣ Complex skin disorder ‣ Loss of pigment cells ‣ 0.5-2 % ‣ No cure or effective treatment Wikipedia. The Free Encyclopedia Wikipedia. The Free Encyclopedia söndag den 7 november 2010
Different hypotheses Autoimmune hypothesis IgG accumulation Biochemichal -‑ O 2 hypothesis Oxidative stress söndag den 7 november 2010
Different hypotheses Autoimmune hypothesis Protein A IgG protease (Z-domain) Proteins masking the autoimmune response IgG accumulation Biochemichal -‑ O 2 hypothesis Oxidative stress söndag den 7 november 2010
Different hypotheses Autoimmune hypothesis Protein A IgG protease (Z-domain) Proteins masking the autoimmune response IgG accumulation Biochemichal -‑ O 2 hypothesis + Oxidative stress Genes involved in regulation of melanogenesis söndag den 7 november 2010
Different hypotheses Autoimmune hypothesis Protein A IgG protease (Z-domain) Proteins masking the autoimmune response IgG accumulation Biochemichal -‑ bFGF SOD yCCS O 2 hypothesis + Tyrosinase MITF Oxidative stress Genes involved in regulation of melanogenesis söndag den 7 november 2010
Different hypotheses Autoimmune hypothesis Protein A IgG protease (Z-domain) Proteins masking the autoimmune response IgG accumulation Biochemichal -‑ bFGF SOD yCCS O 2 hypothesis + Tyrosinase ( ) ( ) MITF Oxidative stress Genes involved in regulation of melanogenesis söndag den 7 november 2010
Vision söndag den 7 november 2010
Vision 1 Bacteria applied söndag den 7 november 2010
Vision 1 Bacteria applied 2 Proteins secreted 2 söndag den 7 november 2010
Vision 1 Bacteria applied 2 Proteins secreted 2 3 3 Internalization söndag den 7 november 2010
Vision 1 Bacteria applied 2 Proteins secreted 2 4 Re-pigmentation 3 3 Internalization söndag den 7 november 2010
Skin penetration ‣ Effective barrier ‣ Needle-free approach ‣ Cell-penetrating peptides (10-20 a.a.) ‣ 3 types tested ‣ Able to carry cargo into cells/skin CPP Cargo Huang, et al . (2010) söndag den 7 november 2010
Wet-lab approach 2 Expression 1 Cloning 3 Purification 4 Activity assays 5 Skin & cell penetration söndag den 7 november 2010
Cloning 1 Cloning 2 Expression 3 Purification 4 Activity 5 Skin & cell penetration söndag den 7 november 2010
Cloning 1 Cloning 2 Expression 3 Purification 4 Activity 5 Skin & cell penetration söndag den 7 november 2010
Cloning 1 Cloning 2 Expression 3 Purification 4 Activity 5 Skin & cell penetration söndag den 7 november 2010
Cloning 1 Cloning 2 Expression 3 Purification 4 Activity 5 Skin & cell penetration × 3 5 söndag den 7 november 2010
Cloning 1 Cloning 2 Expression 3 Purification 4 Activity 5 Skin & cell penetration + × × 3 5 5 3 söndag den 7 november 2010
Cloning 1 Cloning 2 Expression + 3 Purification 4 Activity 5 Skin & cell penetration ( + ) × × 2 3 5 5 3 söndag den 7 november 2010
Cloning 7 × 1 Cloning + 2 Expression + 3 Purification 4 Activity 5 Skin & cell penetration ( + ) × × 2 3 5 5 3 söndag den 7 november 2010
Cloning 1 Cloning 2 Expression 3 Purification 4 Activity 5 Skin & cell penetration söndag den 7 november 2010
Protein expression 1 Cloning ‣ Low/no pEX expression ‣ Experimental bottleneck 2 Expression 3 Purification BioBrick pEX P tac Commercially available 4 Activity expression vector P tac expression vector 0 h 1 h 2 h 3 h 0 h 1 h 2 h 3 h λ λ 5 Skin & cell penetration bFGF + GST bFGF + 6x His söndag den 7 november 2010
Protein purification 1 Cloning ‣ Nickel resin columns 2 Expression ‣ 6x His tag 3 Purification 4 Activity 5 Skin & cell penetration söndag den 7 november 2010
Protein activity SOD activity IgG protease activity 1 Cloning ‣ Digestion of IgG ‣ SOD determination kit secondary antibodies (Sigma-Aldrich) 2 Expression + substr. 3 Purification 4 Activity - O 2 conversion IgG digestion 5 Skin & cell penetration söndag den 7 november 2010
Skin & cell penetration 1 Cloning ‣ Immunohistochemistry 2 Expression ‣ α -His-tag antibodies Fixation 3 Purification ‣ Cultured cells ‣ Keratinocytes 4 Activity ‣ Melanocytes 5 Skin & cell ‣ Skin penetration + Cross-section ‣ Cultured artificial (human) ‣ Extracted live (mouse) söndag den 7 november 2010
Modeling Aim ‣ Model for safe induction of protein expression ‣ Toy model based on lac operon ‣ Well studied ‣ LacI regulated expression vector söndag den 7 november 2010
Modeling Expression vector lac operon mRNA mRNA Perm. β -gal Repressor Internal External Allolactose lactose lactose söndag den 7 november 2010
Modeling Expression vector lac operon mRNA mRNA Perm. β -gal Repressor Internal External Allolactose lactose lactose Ahmazadeh, et al . (2005) söndag den 7 november 2010
Future prospects ‣ Transfer to Lactobacillus ‣ Allows for secretion ‣ ”Skin-friendly” ‣ Other applications söndag den 7 november 2010
Future prospects Lactobacillus Escherichia coli Expr. vector ‣ Transfer to Lactobacillus ‣ Allows for secretion ‣ ”Skin-friendly” ‣ Other applications söndag den 7 november 2010
Our special thanks to Our supervisors Elisabeth Haggård Robert Daniels Gunnar von Heijne ...and Sridhar Mandali Jaroslav Belotserkovsky Richard Odegrip Sergey Surkov ...and everyone else who made this project possible Our sponsors Faculty ¡of ¡Science, Swedish ¡Vi:ligo ¡Associa:on Stockholm ¡University Thermo ¡Fisher ¡Scien:fic Sigma-‑Aldrich Inspira:onal ¡SoBware ¡for ¡Biologists söndag den 7 november 2010
Recommend
More recommend
