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

My 10-minute presentation… I’m super-serious! (Hopefully). This presentation will include amazing topics like: The Latest in Tweezers Technology Biological Advancements And more…. By And KochLab. Thank You!. KochLab Guys Steve Koch – Lab Don Larry Herskowitz – programmer guy

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

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  1. My 10-minute presentation… I’m super-serious! (Hopefully) • This presentation will include • amazing topics like: • The Latest in Tweezers Technology • Biological Advancements • And more… By And KochLab

  2. Thank You! KochLab Guys • Steve Koch – Lab Don • Larry Herskowitz – programmer guy • Andy Maloney – optics Guy • Linh Le –undergrad guy • NasManole – undergrad guy • Brandon Beck – former biology guy Osley Lab People • Mary Ann Osley – head honcho • Kelly Trujillo – mentor guy • Another Awesome Person • Susan Atlas – got us a kick ass grant

  3. Last Time… THE ALL-PURPOSE KOCHBOOK • Recap: • Prototype optical tweezers • Chromatin biology • Prelude to Shotgun DNA Mapping OF KOCHING • The American Science Classic • Revised and Expanded • With Over 4,500 Experiments • And 1,000 Informative Illustrations by ANTHONY SALVAGNO and KOCHLAB

  4. Optical Tweezer Progress Before After • Red (690nm) laser • Built from components • More adaptable system • Unsuccessful trapping  • Green (532nm) Laser • Unstable Beam • Successful Trapping 

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

  6. Biological Progress (Before) • 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 (not same picture as earlier)

  7. Biological Progress (After) • New Aims: • Shotgun DNA Mapping • Unzipping of RNA during elongation • Shotgun Chromatin Mapping • Telomere Analysis • ALL UTILIZING OT!

  8. F F Shotgun DNA Mapping

  9. Extraction of DNA Steps: Destroy yeast and take DNA Use RE to cut DNA into fragments Use plasmids to create clones Attach DNA to tethering construct Unzip and match to library plasmid linearized plasmid Restriction enzyme Combine for tons of DNA Restriction enzyme chromosome Genomic DNA Fragments

  10. Unzipping of RNA Pol II • RNA Pol II is antisymetric • can tell difference between sense and antisense. • Once force signature is established, we can input into library for SCM. Reassembled Nucleosomes RNA Pol II promoter cryptic promoter Transcription • Other Studies: • Promoter-proximal stalling • Initiation complex analysis • Understanding of stalling and termination behavior • More questions out there

  11. Shotgun Chromatin Mapping In principle, very similar to SDM: Unzip non-naked DNA Pop off nucleosomes and polymerases Allow DNA to reaneal Unzip naked DNA Determine locations of popped proteins on DNA fragment Determine location of fragment within genome using SDM library (Larry) Perform several times to get precise locations of nucleosomes and polymerases High throughput for human genome adaptation

  12. 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

  13. Thanks for listening Kochlab.blogspot.com Openwetware.org/wiki/User:Anthony_Salvagno Openwetware.org/wiki/Koch_Lab

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