My 10-minute presentation… I’m super-serious! (Hopefully)

By

And KochLab

This presentation will include amazing topics like:•The Latest in Tweezers Technology•Biological Advancements•And more…

Thank You!KochLab Guys• Steve Koch – Lab Don• Larry Herskowitz – programmer

guy• Andy Maloney – optics Guy• Linh Le –undergrad guy• Nas Manole – undergrad guy• Brandon Beck – former biology

guy• Diego Ramallo – KochLab alumni

Osley Lab People• Mary Ann Osley – head honcho• Kelly Trujillo – mentor guy

Another Awesome Person• Susan Atlas – cool kinesin project

Last Time…

• The American Science Classic• Revised and Expanded• With Over 4,500 Experiments• And 1,000 Informative

Illustrations

THE ALL-PURPOSE KOCHBOOK

OFKOCHING

by ANTHONY SALVAGNO and KOCHLAB

Recap:•Prototype optical tweezers•Chromatin biology•Prelude to Shotgun DNA Mapping

Before After•Green (532nm) Laser•Unstable Beam•Successful Trapping

•Red (690nm) laser• Built from components•More adaptable system•Unsuccessful trapping

Optical Tweezer Progress

Biological Background•DNA is important to a lot of processes in the cell•Histones compact DNA for storage and regulation•Histone remodification needed for control of cellular processes like:• Protein synthesis• replication

Biological Progress (Before)

(not same picture as earlier)

•Focus on histone remodeling using optical tweezers•Shundrovsky et al. proved nucleosome location can be found (resolution of 3bp)•We want to map nucleosome locations throughout a gene•Use PHO5 gene because well documented locations (proof of principle)•Getting Chromatin from yeast cells prove to be very difficult

Biological Progress (After)

New Aims:•Shotgun DNA Mapping•Unzipping of RNA during elongation•Shotgun Chromatin Mapping•Telomere Analysis

ALL UTILIZING OT!

Shotgun DNA Mapping

F

F

Extraction of DNA

plasmidlinearized plasmidRestriction

enzyme

Restriction enzymechromosome

Genomic DNA Fragments

Combine for tons of DNA

Steps:1. Destroy yeast and take DNA2. Use RE to cut DNA into fragments3. Use plasmids to create clones4. Attach DNA to tethering construct5. Unzip and match to library

Unzipping of RNA Pol II

RNA Pol II

Transcription

Reassembled Nucleosomes

promoter

crypticpromoter

RNA Pol II is antisymetric • can tell difference between sense and

antisense.Once force signature is established, we can input into library for SCM.

Other Studies:•Promoter-proximal stalling•Initiation complex analysis•Understanding of stalling and termination behavior•More questions out there

Shotgun Chromatin MappingIn principle, very similar to SDM:1. Unzip non-naked DNA2. Pop off nucleosomes and

polymerases3. Allow DNA to reaneal 4. Unzip naked DNA5. Determine locations of

popped proteins on DNA fragment

6. Determine location of fragment within genome using SDM library (Larry)

7. Perform several times to get precise locations of nucleosomes and polymerases High throughput for human genome adaptation

Future Experiments

• Utilize SDM/SCM for cool stuff– Detection of inversions, deletions, …– Alternative Splicing

• Telomere Mapping– Cancer research related– Aging related– Loaded with repeat sequences– Lots can be done with OT

Thanks for listening

Kochlab.blogspot.com

Openwetware.org/wiki/User:Anthony_SalvagnoOpenwetware.org/wiki/Koch_Lab