The Joy Of Koching 6/13/08
-
Upload
anthony-salvagno -
Category
Education
-
view
528 -
download
0
description
Transcript of The Joy Of Koching 6/13/08
The American Science ClassicRevised and ExpandedWith Over 4,500 ExperimentsAnd 1,000 Informative Illustrations
THE ALL-PURPOSE KOCHBOOK
OFKOCHING
by ANTHONY SALVAGNO and KOCHLAB
Let us give thanks• Kochlab Nation
– Steve Koch (founder and godfather)
– Larry Herskowitz– Patrick Jurney– Linh N Le– Diego Ramallo-Pardo– Nas Manole– Caleb Morse– Adam Wise (NSMS)
• The VIP List– Osley Lab– Lopez Lab– Nick Carroll– Chris Lorenz– Richard Yeh– Karen Adelman
• The Beautiful People
Better Understanding of Cancer on the “Nano” Scale
Modeling Methods• Molecular
Dynamics• Monte Carlo
Simulations
Physical Methods• Optical
Tweezers• Microfluidics• Nanomanipul
ation (future)
Biological Methods• Chromatin
Extraction• DNA Molecule
Tethering
Bread Making 101
• Extracting Chromatin from living yeast cells• Tethering extracted Chromatin to slide
Overview of process:Part 1: Part 2:
Korber et al. MCB . (2004). 24: 10965-10974
Preheat the Oven to 350°
1st knead the dough…
• Need Pho5 plasmid– Contains well documented nucleosome locations– Highly understood
Cell Wall
Mock yeast cellFrom Wikimedia Commons
Nucleus • To digest chromatin one needs access to nucleus– 1st attempts involved
creation of spheroplasts– Current attempts use
direct contact with nucleus
…next allow dough to rise • After extraction, tethering commences– Chromatin needs to be prepared in a tethering
construct (dig labeling on one end and biotin labeling on the other).
Above: Tethering Constuct (Steve Koch)
Left: Side profile of tethered particles ready for experimentation. (Diego Ramallo-Pardo)
Put it in the oven…• Tethered Particle Motion
– Use “macro” microscopic reporters such as QDs or microbeads– Watch what happens
• A simple experiment: Does size matter?– Comparing the motion of DNA when fixed to QDS and microbeads
TPM of 3 microbeads
Courtesy of Salvagno et al (2008)
… and bake for 30-40 min
• Optical Tweezers– Allow manipulation of
DNA tethers
• Microfluidics– Allow automation of
tethering process– Provides backbone to
many future experiments
The Tools of the Trade
Tweezing courtesy of Dr. Koch Microchannel by AS
Make sure to check frequently.
• Building OT– Need laser– Beam steering– Data Acquisition– Microscope
Simple Design by Block and Neuman (2004)
Place on wire rack to cool.• Building Microchannels
– Double Stick Tape Method– PDMS 1.0
• Using Photoresist– Greatly improves precision of channel making– Can create all types of different shapes and sizes for use in various
experiments
Mask Design by Patrick Jurney
Presentation is important
Simulations: • Thermodynamic calculations of naked DNA
unzipping• Monte Carlo simulations of RNA Polymerase– Transcription on chromatin
• Molecular Dynamics of protein-DNA interactions– Histone-DNA and RNA Pol II-DNA– Atomistic MD for microsecond timescale– Coarse-grained MD for larger scales
More Recipes…
• Shotgun Chromatin Mapping– Create shotgun chromatin
fragments using I-PpoI– Tether fragments– Unzip fragments
…and even more!
• $6 Million (Man) Tweezers– Beam Steering
• AOD• Mirror Galvanometer
– Better Data Acquisition– Feedback
• Micro- (and nano-) fluidic experiments– PDMS stamping– Flow experiments
• Simulations– Combination of atomistic and CG MD
The End