HokkaidoU2011.igem.org/files/presentation/HokkaidoU_Japan.pdfBiofusion standerd takes… BioBrick...
Transcript of HokkaidoU2011.igem.org/files/presentation/HokkaidoU_Japan.pdfBiofusion standerd takes… BioBrick...
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100 nm
3 um
1 um E.coli
= 2 nm
80 nm
Protein delivery into eukaryotic cells by type III secretion machines Jorge E. Galan and Hans Wolf-Watz, 2006, Nature 444: 567-573
About T3SS
Bacterial cytosol
Target cell cytosol
Target cell membrane
Inner membrane
Outer membrane
pSB1C3
E. coli K-12 [SPI-2/T3Signal-GFP/RFP]
pBAC SPI2
secretion apparatus
target cell
pSB1C3
Promoter RBS T3S signal GFP Double
terminator RFP
reporter RFP
reporter T3S signal GFP
Fused protein
GFP
GFP
Gene Construct
Result
Merge GFP
RFP phase-contrast
Objectives for iGEM 2011
I. Making injection assay easier by using onion cells sheets
II. Screening what proteins can be injected by T3SS
III. Seeking a method for verifying the injection of proteins
Animal cells Onion cells
Difficult to prepare
Weak against bacteria
Easy to prepare
Tolerant against bacteria
1 2 3
4 5
6 7
Result
A B C
Objectives for iGEM 2011
I. Making injection assay easier by using onion cells sheets
II. Screening what proteins can be injected by T3SS
III. Seeking a method for verifying the injection of proteins
Wish to clone various proteins into…
HERE
S N P SlrP sig.
TetR
RBS E N X
Double Terminator
Biofusion standerd takes…
BioBrick
Biofusion Prefix primer
Biofusion Suffix primer
Too much cost and time!
E N X S N P
What should we do?
Four requirements for cloning system
I. Inserting various kinds of BioBrick parts into another Biobrick using a SAME primer set for PCR.
II. Making it sure that the part is to be inserted in the correct direction.
III. Producing a fusion protein.
IV. Resulting construct to retain BioBrick properties.
Objectives for iGEM 2011
I. Making injection assay easier by using onion cells sheets
II. Screening what proteins can be injected by T3SS
III. Seeking a method for verifying the injection of proteins
Glycogen Synthase Kinase 3β
GSK tag and location
Bacterial cytosol
Eukaryotic cytosol
Phosphorylated NO YES
unmodified YES NO
Location
GSK Tag state
Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang
Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by
Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57.
GSK tag and antibodies
Anti phosphorylated
GSK Anti GSK
Phosphorylated Recognizes Recognizes
unmodified - Recognizes
Atibodies
GSK Tag state
Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang
Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by
Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57.
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Acknowledgement
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