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Tracking evolution of gene regulatory circuits

Tracking evolution of gene regulatory circuits. Serdar Turkarslan. 11.02.09. Scope. To understand evolution of gene regulatory circuits -- via expansion of general transcription factors -- in response to changing environment. Methodology. Survey of fitness landscape

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Tracking evolution of gene regulatory circuits

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  1. Tracking evolution of gene regulatory circuits • Serdar Turkarslan 11.02.09

  2. Scope • To understand evolution of gene regulatory circuits -- via expansion of general transcription factors -- in response to changing environment

  3. Methodology • Survey of fitness landscape • Sub-functionalizations • Complementation • Phylogeny • Directed Selection • Single • Variant Library • Competition • Co-optimization • Systems Analysis

  4. Determination of fitness landscape Knockouts Environment … … … … … pH 3.0 5.0 7.0 9.0 Metal (Cu) 0.40 mM 0.80 mM 1. 00 mM Salt 2.5 M 3.0 M 3.5 M 4.0 M 5.0 M Temp. 25 C 37 C 42 C TFB a, b, c, d, e TBP a, c, d

  5. Surveying fitness landscape 1. Identify fitness landscape for single perturbations 2. Determine subset combinatorial perturbations that will yield physiologically relevant maximal fitness changes

  6. Cu Perturbation

  7. Temperature (42ºC) Perturbation 42ºC 2.5M Salt 42ºC CM 42ºC 1.0 mM Cu

  8. Competition of GTF gene knockouts Growth Competition qPCR Analysis

  9. NextGen Sequencing

  10. NextGen Sequencing tfbC qPCR tbpC qPCR

  11. tfbD-tfbE gene chimera

  12. PtfbE PtfbD TFBd* TFBd* tfbD-E chimera tfbD/tfbE promoter constructs ∆-ura3 Low Temperature ∆-tfbD ∆ -tfbE Complementation

  13. How about other orthologs?

  14. Next • Survey more perturbations • Select combinatorial perturbations • Continuous Competition experiments in 16-plex chemostat • Deep sequencing analysis • Complementation with other GTFs

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