SLIDE 1 Our lab
Yeshiva University
Spatially encoding temporal information: using diffusional escape of periplasmic reporter proteins as a clock.
Faige Seligman, Juliet Meir, Raphael Huntley
- Prof. Michael Machczynski, Prof. Neer Asherie
SLIDE 2
1 2 3 1 2 3
Cytoplasm Periplasm Media
SLIDE 3 The Set-up:
cytoplasm periplasm
SLIDE 4
TAT and Sec Pathways
Unfolded While In Cytoplasm
TAT
More Hydrophobic Basic Carboxyl-terminus Twin Arginine Dipeptide in Amino-terminus Already Folded In Cytoplasm Shorter Leader Sequence Neutral Carboxyl-terminus Less Hydrophobic Temperature Independent Temperature Dependent
Sec
SLIDE 5 Periplasmic Leader Sequences
Name GFP-SsrA FACS mean fluorescence (WT) PhoA Activity Export Pathway YcbK 82 3 TAT YahJ 100 3 TAT+Sec YcdB (Y) 123 6 TAT+Sec AmiC (A) 36 18 TAT+Sec MdoG (M) 15 165 TAT+Sec FhuD (F) 6 256 TAT+Sec
Danielle Tullman-Ercek et al., J Biol Chem. 282, 8309-8316 (2007).
SLIDE 6 Standard Suffix Standard Prefix (Short)
Leader Sequence TAC TAG TAG CGG CCG CTG CAG GAA GAA AC T
5’ 3’
GTT TCT TCG AAT TCG CGG CCG CTT CTAG ATG TTA CTA GAG
BioBrick Scar:
LEU LEU GLU …CCG CTT CTAGAG
Prefix of Next Adjoining Part 3’ 5’
SLIDE 7 Building Leader Sequence BioBricks
http://www.neb.com/nebecomm/products/productE0546.asp .1 .2 .3 .5 kbp
LS PCR Products
SLIDE 8 Final Construct:
Gene of Final Construct is Expressed LS/Protein Travels to Membrane LS is Removed and Protein is Exported Through TAT or Sec
RBS: BioBrick BBa_B0030. Promoter: BioBrick BBa_I712074
SLIDE 9 Our Genes of Interest and Results
.1 .2 .3 .5 kbp 1.0 10.0
Human Eye
Success
BBa_E1010
RFP
Be Successful
Lab
Cytoslac
Weight
Express
Express RFP PCR Product
SLIDE 10 Braun’s lipoprotein Periplasmic space
http://en.wikipedia.org/wiki/File:Gram_negative_cell_wall.svg
SLIDE 11 gene
Cam
3’ 3’
Cam
Red/ET system: γ, α (exo) & β
exonuclease Redγ gene: Inhibit RecBCD Redβ gene: Annealing protein
Cam
3’ 3’
Cam
3’ 3’
Redα gene: 5 ’ 3’ exonuclease
SLIDE 12 SDS/PAGE
2hr 12hr
Red/ET (Amp) Arabinose 2hr 4hr 6hr 8hr Competent cells
Amp/Cam Resistant Cam Resistant
Transformation
SLIDE 13
2 hr 8hr 1.0Kb gene
Cam
0.5Kb 1,000 base pairs 1,300 base pairs 1.3Kb
SLIDE 14
Petri Dish Reporter Time: 0 hours Time: x hours Time: x + y hours
SLIDE 15
Dry Run: To monitor diffusion of dyes. Test
for
the
choice
of
dye
SLIDE 16
Procedure of Diffusion Analysis
Full
picture
of
agar
plate
with
dye
SLIDE 17 4
hours
2
hours
Variables
Tested:
‐Thickness:
1x
and
2x
agar
plates.
10 20 30 40 50 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
4 Hours 2 Hours
Position (cm)
Intensity
SLIDE 18 Modeling the Data Diffusion Equation (1D): Solution:
Intensity
(arb.)
2 hours
Position (cm)
10 20 30 40 50 0.5 1 1.5
Fit Data
SLIDE 19
Experimental Diffusion Coefficients
Dyes and Proteins D
(10−6
cm2/s)
Number of Strips Methyl Red 5±1 7 Cytochrome c 3±1 6
SLIDE 20
1 2 3 1 2 3
Cytoplasm Periplasm Media
Summary (part 1)
SLIDE 21
Summary (part 2)
Step of project Goal Achievement 1 To export proteins Leader Sequence BioBricks 2 Delete Lpp gene PCR product 3 Develop method of diffusion analysis 1D Diffusion
SLIDE 22 Acknowledgements
We would like to thank:
- Prof. Michael Machczynski
(left) and Prof. Neer Asherie (right) for their help, dedication, and support.
- iGEM for the experience and tools.
- Dr. Henry Kressel and YU for their
financial support
