nsms igert nano cafe 2/12/09
DESCRIPTION
This is a talk I gave at the Nano Cafe in February. It is a quick overview of the things I am doing in KochLab and some of the things we hope to achieve.TRANSCRIPT
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