detection of local protein structures along dna …...present advantages over biological pores...
TRANSCRIPT
![Page 1: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/1.jpg)
Detection of local protein structures along DNA using solid-state nanopores
Stefan Kowalczyk Adam Hall
Cees Dekker
Bremen 29-06-2009
nanopore
RecA-DNA filament
(Nano Letters cover September 2009)
![Page 2: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/2.jpg)
Main topics:
RecA
dsDNA
• Solid-state nanopores for proteins on DNA
• Nuclear pore complex (NPC)
![Page 3: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/3.jpg)
http://sspatel.googlepages.com/nuclearporecomplex
Main topics:
• Solid-state nanopores for proteins on DNA
• Nuclear pore complex (NPC)
![Page 4: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/4.jpg)
Stefan KowalczykMichiel van den HoutGary SkinnerAdam HallRalph SmeetsMeng-Yue WuUlrike ZieseSerge LemayNynke DekkerCees Dekker
Biopolymer translocation through nanopores
![Page 5: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/5.jpg)
Outline
• Brief motivation
• Fabrication of solid-state nanopores
• Translocation of dsDNA through solid-state pores
• Translocation of RNA
• Translocation of protein-coated DNA
• Very first data on artificial nuclear pores
![Page 6: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/6.jpg)
http://www.chemsoc.org/exemplarchem/entries/2002/Tim_Smith/transp http://www.scripps.edu/~stoffler/proj/NPC/npc.html
Nanopores in biology:• ion channels
• nuclear pore complexes
• viral infection
• protein secretion
• bacterial gene transfer
• etc etc
![Page 7: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/7.jpg)
Dreams of nanopore-based DNA sequencing
![Page 8: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/8.jpg)
Nanopores for biotechnology and biophysics: ssDNA/RNA through α-hemolysin biopores
(Kasianowicz, Branton, Akeson, Deamer, Meller, …)
Blockades of 100 bp poly(dA) through an α-hemolysin membrane protein
from Meller et al. PNAS 97, 1079 (2000)
![Page 9: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/9.jpg)
Solid-state nanopores
present advantages over biological pores within a lipid membrane:
Flexibility in pore diameter and pore lengthHigh stability (temperature, pH, salt, ..)Adjustable surface properties of the pore Allows integration into devices and arrays
![Page 10: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/10.jpg)
SiO2
or SiN nanopores from silicon processing
![Page 11: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/11.jpg)
Fine tuning by ‘glass making’
with a TEM beam True nanometer control of the nanopore size
High-intensity TEM slowly closes the pore, with live imagingStop imaging to ‘freeze’ the geometry for nm-sized pore
5 nm
A. Storm et al, Nature Mater. 2, 537 (2003)
![Page 12: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/12.jpg)
Measuring the ionic current through a nanopore
![Page 13: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/13.jpg)
dsDNA translocation through a 10 nm nanopore
A. Storm et al, Phys. Rev. E71, 051903 (2005)
![Page 14: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/14.jpg)
Rich variety of experimental results for dsDNA
Folding phenomena
Sign reversal of current signal at low salt
τ
~ Lα
1M 0.1M
Power-law length dependence of translocation time
Noise studies, evidence for nanobubbles
![Page 15: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/15.jpg)
Nanopore technique basically applicable to any charged molecule
- polynucleotides
- proteins -
protein-DNA complexes
-
polyelectrolytes
- nanotubes - nanowires -
quantum dots
etc
Three examples of recent research1. double and single strand RNA2. proteins on DNA3. artificial nuclear pore complexes
![Page 16: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/16.jpg)
Comparison of different polynucleotides
G. Skinner, M. van den Hout et al, Nano
Letters (2009)
![Page 17: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/17.jpg)
ds RNA
ss RNA
G. Skinner, M. van den Hout et al, Nano
Letters (2009)
![Page 18: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/18.jpg)
Current blockade amplitudes differ at high fields
dGC
ondu
ctan
ce c
hang
e dG
(nS
)
![Page 19: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/19.jpg)
Translocation of proteins and protein-coated DNA
Screening of local structures along DNA, e.g., proteins, transcription factors, nucleosomes, etc
![Page 20: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/20.jpg)
Our model system: RecA protein on DNA
RecADNA
![Page 21: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/21.jpg)
dsDNA RecA-coated dsDNA
R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
AFM imaging of RecA-coated DNA
![Page 22: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/22.jpg)
Blockades are 15 times bigger than for dsDNA
Consistent with large cross sectiondG => d=8.5 nm
R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
Translocation of RecA-coated double-strand DNA
![Page 23: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/23.jpg)
Translocation of RecA-coated double-strand DNA
RecA + DNA RecA only
![Page 24: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/24.jpg)
3) Two different regimes (constant and exponential event rate vs. voltage)
1) Dwell times of bare DNAand fully RecA-coated DNAare equal within error(surprisingly?)
2) Poissonian
process (“no memory”)
R. Smeets, S. Kowalczyk, et al, Nano Lett. (2008)
Some lessons learned:
![Page 25: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/25.jpg)
Next step: “Read out”
DNA along its length
![Page 26: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/26.jpg)
Local patches of RecA protein along DNA
S. Kowalczyk, et al, submitted
Height scale [0-2 nm]
![Page 27: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/27.jpg)
translocation data conductance histogram
(dG = dI/V)
Local patches of RecA protein along DNA
S. Kowalczyk, et al, submitted
![Page 28: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/28.jpg)
S. Kowalczyk, et al, submitted
![Page 29: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/29.jpg)
![Page 30: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/30.jpg)
What is the best resolution we can achieve?
baseline
DNA
RecA
![Page 31: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/31.jpg)
Total translocation time is inversely proportional with voltage
S. Kowalczyk, et al, submitted
![Page 32: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/32.jpg)
Resolution of protein along DNA: ~ 60 bp(probably even better in a very recent data set, ~ 30 bp)
S. Kowalczyk, et al, submitted
![Page 33: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/33.jpg)
VV
The Optical Tweezer-Nanopore System
Same trick with optical tweezers?
A. Hall, et al
![Page 34: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/34.jpg)
20
10
0
-10
100
50
0
-50
The Optical Tweezer-Nanopore System
A. Hall, et al
![Page 35: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/35.jpg)
Applying RecA to the Hybrid System
1M KCl
A. Hall, et al
![Page 36: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/36.jpg)
A. Hall, et al
First data on captured partly-RecA
coated DNA
Under investigation…
![Page 37: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/37.jpg)
Salt dependence of RecA-DNA translocations (preliminary data)
“Crossover”
~0.35M KCl; in agreement with R. Smeets, et al, Nano
Letters, 2006
dsDNA
RecA-DNA
dsDNA
RecA-DNA
![Page 38: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/38.jpg)
Salt dependence of RecA-DNA translocations (preliminary data)
0.2M KCl
• Current increases from DNA• Current decreases from RecA-DNA
![Page 39: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/39.jpg)
http://sspatel.googlepages.com/nuclearporecomplex
Main topics:
• Solid-state nanopores for proteins on DNA
• Nuclear pore complex (NPC)
![Page 40: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/40.jpg)
The cell as a collection of
protein machines
The only way to get from the nucleusto the cytoplasm is through a nuclear pore complex
![Page 41: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/41.jpg)
Use solid-state nanopores as a chassis to build biomimetic artificial nuclear pore complexes
http://sspatel.googlepages.com/nuclearporecomplex
in collaboration with Roderick Lim and Ueli Aebi (Basel)
![Page 42: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/42.jpg)
![Page 43: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/43.jpg)
Small Molecules can diffuse freely through the Nuclear Pore,Larger molecules require active transport,
Cartoon Biology:
![Page 44: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/44.jpg)
F. Alber, et al,Nature 450, 695-701, 2007
![Page 45: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/45.jpg)
F. Alber, et al,Nature 450, 695-701, 2007
![Page 46: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/46.jpg)
Roderick Lim, et al, Science (2007)
“Selective gating”
/ “virtual gating”
![Page 47: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/47.jpg)
Recent simultations
from Klaus Schulten’s
group (Urbana)
L. Miao and K. Schulten, Structure, 17, (2009)
MD evidence thatFG-nups
form
brushes
![Page 48: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/48.jpg)
Building a “minimalistic NPC”…
Thanks to Roderick Lim and Larisa Kapinos-Schneider! (Basel University)
…
starts withprotein purification
![Page 49: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/49.jpg)
Next task: chemistry thinking…How to attach those FG-proteins to the SiN…?
Our hero cross-linker:
NH2- -SH
![Page 50: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/50.jpg)
Transport of Importin-Beta throughbare and modified nanopores
FG-nups
![Page 51: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/51.jpg)
TEM images of the same 40 nm nanoporeBefore (a) and after (b,c,d) attachment of FG-nups
![Page 52: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/52.jpg)
Power spectral analysis before and after attachment:
![Page 53: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/53.jpg)
Example traces of Importin-Beta translocations through a bare and modified nanopore
ImpB through a bare pore ImpB through a modified pore
![Page 54: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/54.jpg)
Translocation time histograms for ImpB translocations through bare and modified nanopore
<Tdwell
> = 0.12 ±
0.02 ms <Tdwell > = 2.7 ±
0.5 ms
![Page 55: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/55.jpg)
Some indications/confirmations that the FG-nups bind to our solid-state nanopore, and that we can do
transport measurements:
•Ellipsometry
data indicates extra layers of expected thickness•TEM images show some “stuff”
in the pore that’s very sensitive to the electron beam•Power spectra before and after are clearly different•Open pore current decreases systematically upon binding of nups
(dependence of pore size and type of nups)
•Translocation data show 20-fold (!) increase in translocation time for Importin-Beta for bare vs. FG-
nanopore, with equal event amplitudes
•More experiments on the way..
![Page 56: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/56.jpg)
Summing up:
We have used solid-state nanopores for :-
variety of experiments on dsDNA
-
experiments on ssRNA and dsRNA-
experiments on RecA proteins along DNA
-
biomimetic nuclear pore complexes
![Page 57: Detection of local protein structures along DNA …...present advantages over biological pores within a lipid membrane: 9 Flexibility in pore diameter and pore length 9 High stability](https://reader033.vdocuments.us/reader033/viewer/2022050111/5f48de3534a12737275adf3b/html5/thumbnails/57.jpg)
One-line summary:
Solid state nanopores are versatile new probes for biophysics
postdoc openings!!
15 faculty openings at Delft as well !!