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Geoffrey Lovely, Sungwook Woo, and David Schwab, with help of Stephanie Johnson

To Loop, or Not to Loop, That is the Question. Geoffrey Lovely, Sungwook Woo, and David Schwab, with help of Stephanie Johnson. Why should you care about Looping?. Transcriptional Regulation - Difference Between Life and Death VDJ Recombination - Difference Between Life and Death

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Geoffrey Lovely, Sungwook Woo, and David Schwab, with help of Stephanie Johnson

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  1. To Loop, or Not to Loop, That is the Question. Geoffrey Lovely, Sungwook Woo, and David Schwab, with help of Stephanie Johnson

  2. Why should you care about Looping? • Transcriptional Regulation - Difference Between Life and Death • VDJ Recombination - Difference Between Life and Death • Evolution - Difference Between Life and Death

  3. LAC OPERON PLac Lac Z Lac A LacI O3 O1 O2 Lac Y 1080 92 401 2671 1251 609 bps Lac I ( Repressor) Operators (O1> O3> O2) Lac Z(ß-Galactosidase) Lac Y(Permease) Lac A (Transacetylase) Promoter (PI and Plac) Oehler et al. 1994

  4. Lac Repressor To Simplify = Tetramer bound to two operators Swigon et al. 2006

  5. Lac Operon in the Presence of Glucose Glucose LacI O3 O1 O2 mRNA Tetramer of Repressor Protein Monomer of Repressor • 1) In the Presence of Glucose Lac Repressor is produced • 2) Lac Repressor binds to operator halts PLac expression Note: In solution the Repressor forms tetramers (Ruben et al. 1997)

  6. O3 O1 O2 All Possible Binding States One Tetramer O3 O1 O2 O3 O1 O2 Two Tetramers O3 O1 O2 O3 O1 O2 O3 O1 O2 Three Tetramers O3 O1 O2

  7. All Possible Looped States of Lac Repressor One Tetramer Two Tetramers

  8. Experimental Scheme We use beads (~0.5 mm) to observe single DNA molecules. Lin Han’s thesis

  9. Experimental Scheme Lac The effective length of the DNA strand will get shorter when looped. Hence, the lateral scope of the movement of the bead will get smaller.  The movement of each bead indicates the state of each single DNA molecule!

  10. Experimental Scheme Glass Slide Output Input DoubleSided Tape Cover Slip

  11. Experimental Scheme

  12. Results & Discussion Raw data RMS

  13. Results & Discussion Raw data RMS

  14. Concentration Dependence 1 Probability 0 1X10-12 1X10-8 1X10-4 Concentration (M)

  15. Histograms of RMS Data No Lac 100 fM O1-O3 O1-O2 No Loop 100 pM 100 nM

  16. Conclusion Future work • Data analysis: Gaussian filter, Hidden Markov • Move/delete operators, relative phasing • Measure looping free energies • More detailed concentration dependence • Kinetics of transitions

  17. Results & Discussion

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