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