Yeshiva University Our lab Media 2 3 Cytoplasm 1 Periplasm 1 - PowerPoint PPT Presentation

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 Yeshiva University Our lab

Media 2 3 Cytoplasm 1 Periplasm 1 2 3

The Set-up: periplasm cytoplasm

TAT and Sec Pathways Twin Arginine Dipeptide Shorter Leader Sequence in Amino-terminus More Hydrophobic Less Hydrophobic Basic Carboxyl-terminus Neutral Carboxyl-terminus TAT Sec Temperature Independent Temperature Dependent Already Folded In Cytoplasm Unfolded While In Cytoplasm

Periplasmic Leader Sequences Name GFP-SsrA FACS mean PhoA Activity Export Pathway fluorescence ( WT) 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 6 256 TAT+Sec FhuD (F) Danielle Tullman-Ercek et al., J Biol Chem. 282 , 8309-8316 (2007).

Standard Prefix (Short) 5’ GTT TCT TCG AAT TCG CGG CCG CTT CTAG Standard Suffix ATG Leader Sequence T TAC TAG TAG CGG CCG CTG CAG GAA GAA AC 3’ Prefix of Next Adjoining Part …CCG CTT CTAGAG 5’ 3’ BioBrick Scar: TTA CTA GAG LEU LEU GLU

Building Leader Sequence BioBricks LS PCR Products kbp .5 .3 .2 .1 http://www.neb.com/nebecomm/products/productE0546.asp

Final Construct: Gene of Final Construct is LS/Protein Travels to LS is Removed and Protein is Expressed Membrane Exported Through TAT or Sec RBS: BioBrick BBa_B0030. Promoter: BioBrick BBa_I712074

Our Genes of Interest and Results RFP PCR Product kbp • Visible To The • Incorrect Human Eye 10.0 Weight • History Of RFP Success • Failed To • BioBrick 1.0 Express BBa_E1010 .5 .3 • Very Likely To .2 • Failed To Be Successful Cytoslac Express • Utilized In Our Lab .1

Periplasmic space Braun’s lipoprotein http://en.wikipedia.org/wiki/File:Gram_negative_cell_wall.svg

gene Cam Cam 3’ 3’ Red/ET system: γ , α (exo) & β Red β gene: Red α gene: Annealing Red γ gene: 5 ’ 3’ protein exonuclease Inhibit RecBCD 3’ Cam 3’ Cam 3’ 3’ exonuclease

Arabinose 4hr 2hr 6hr 8hr Red/ET (Amp) Competent cells Transformation SDS/PAGE 2hr 12hr Cam Amp/Cam Resistant Resistant

Cam gene 1,300 base pairs 1,000 base pairs 2 hr 8hr 1.3Kb 1.0Kb 0.5Kb

Time: 0 hours Petri Dish Reporter Time: x hours Time: x + y hours

Dry Run: To monitor diffusion of dyes. Test
for
the

 choice
of
dye


Procedure of Diffusion Analysis Full
picture
of
agar
plate
with
dye



2
hours
 50 40 30 4 Hours Intensity 20 2 Hours 10 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 -10 Position (cm) 4
hours
 Variables
Tested:
 ‐Thickness:
1x
and
2x

 agar
plates.


Modeling the Data 2 hours 50 Diffusion Equation (1D): 40 Intensity
(arb.)
 30 Fit 20 Data 10 0 0 0.5 1 1.5 -10 Position (cm) Solution:

Experimental Diffusion Coefficients D
(10 − 6 
cm 2 /s)
 Dyes and Proteins Number of Strips Methyl Red 5±1 7 Cytochrome c 3±1 6

Summary (part 1) Media 2 3 Cytoplasm 1 Periplasm 1 2 3

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 1D Diffusion diffusion analysis

Acknowledgements • iGEM for the experience and tools. • Dr. Henry Kressel and YU for their financial support We would like to thank: • Prof. Michael Machczynski (left) and Prof. Neer Asherie (right) for their help, dedication, and support.