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¿Podemos diseñar sistemas de expresión que respondan a señales ambientales predeterminadas?

¿Podemos diseñar sistemas de expresión que respondan a señales ambientales predeterminadas? Reguladores a la carta. The transcriptional regulator XylR. B interdomain. 211. 233. D HTH. NH 2. C (activation). COOH. A (receptor). ATP binding. Recognition and binding

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¿Podemos diseñar sistemas de expresión que respondan a señales ambientales predeterminadas?

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  1. ¿Podemos diseñar sistemas de expresión que respondan a señales ambientales predeterminadas? Reguladores a la carta

  2. The transcriptional regulator XylR B interdomain 211 233 D HTH NH2 C (activation) COOH A (receptor) ATP binding Recognition and binding of aromatic effectors DNA binding in Pu y Ps ATP hydrolysis Multimerization Contacts with s54

  3. Activation of XylR in response to inducers R A B R C D R XylR inactive XylR active Intramolecular repression Specific A-C interactions Binding to A domain Release of repression

  4. Regulators á la carte: can we change at will the effector specificity of XylR? A B C D

  5. (XylR16-1) DP HHHR (XylR3H) The B domain of XylR: a coiled coil? 208 236 IVDE RYELQTQ VANLRNR LKQYDGQ YYGIG B/Q linker 566 C D N A C 1 211 233 472 514 554

  6. CH3 NH2 Cl CH3 CH3 CH3 CH3 Cl NO2 CH3 Native inducer 3 5 6 9 10 12 2 CH3 2-methyl 3-Cl tol benzene aniline 4-NT naphthalene CH3 m-xileno Suboptimal inducers A B C XylR 4 7 8 11 13 14 OH CH3-(CH2)6-CH3 F phenol 4-FBA 4-ClBA 1-methyl biphenyl octane Non-inducers

  7. 8 9 1 2 3 4 5 6 7 10 11 12 13 14 Effects of 16-1 mutation in the B domain of XylR 10 MAD1, XylR A B 5 0 MAD16-1, XylR16-1 Activity Pu (b-Gal, x 103) A 10 B C 5 0 Inducer compounds

  8. Can we change the effector specificity of XylR? A B C D

  9. Non natural inducers R R XylRactive Generation of regulators á la carte A B C D XylRinactive

  10. Genetic approach: generation of diversity Structural prediction The A domain of XylR 1 211

  11. toluene m-xylene p-xylene CH3 Native inducers CH3 CH3 CH3 CH3 A B C D New inducers XylR CH3 CH3 CH3 NO2 NO2 NO2 2-NT 3-NT 4-NT biphenyl

  12. Reshaping the effector pocket of the A domain R CH3 CH3 R generation of diversity A

  13. Generation of diversity through mutation-prone shuffling of homologous A domains Family of similar DNA sequences 1. Preparation of target DNA 2. Digestion DNase I (30-300 bp) 3. PCR without oligonucleotides 4. PCR with oligonucleotides XylR A domain DmpR A domain TbuT A domain

  14. Shuffling of A domains of XylR-like activators Shuffled A domains Family of similar DNA sequences Genetic screening Selection of new active combinations Pool of A domain variants XylR A domain DmpR A domain TbuT A domain Elimination of Non productive combinations

  15. The genetic tricks npt (km) Po • Positive selection • Negative selection • Visual screening • Phenotypical characterisation sacB Po luxAB Po lacZ Pu

  16. Genetic screening/selection M9 succ Km + new effector NdeI SnaBI A C D Pr pCon918 A domain Shuffling library NdeI/SnaBI Conjugation P. putida KT2440 Po-Km/Po-sacB colony pool ligation E. coli XL1 Cloning vector Growth on plates Sucrose No Km Plasmid extraction Light emission P. putida KT2440 Po-luxAB

  17. 3-nt decanal test assay no inducer no inducer LB+3NT C- C- LB+3NT

  18. XylR 1 XylR 1 XylR 2 XylR 2 XylR 3 XylR 3 XylR 4 XylR 4 XylR 5 XylR 5 XylRwt XylRwt Visualization of effector-specificity changes no inducer m-xylene phenol benzene 2-nt 3-nt 4-nt biphenyl

  19. Sequence analysis of hybrid regulators 220 161-166 V124A 1 XylR1 DmpR XylR F65L XylR2 46-50 XylR3 L184I XylR4 161-166 XylR5

  20. In vivo activity of hybrid regulators XylR1 XylR2 XylR wt 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 8 8 8 6 6 6 4 4 4 2 2 2 0 0 0 fenol benc 4-NT no ind- 4-NT 2-NT benc 3-NT 3-NT m-xil 2-NT 2-NT m-xil fenol fenol 3-NT m-xil benc 4-NT bifenilo bifenilo no ind- no ind- bifenilo m-xileno benceno Promoter activityr Pu (b-Gal, x 103) XylR5 XylR4 XylR3 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 8 8 8 6 6 6 4 4 4 2 2 2 0 0 0 fenol 2-NT 4-NT benc 3-NT 2-NT 3-NT 4-NT m-xil no ind- bifenilo fenol m-xil fenol benc 2-NT 3-NT 4-NT m-xil benc bifenilo no ind- no ind- bifenilo

  21. (Apparent) affinity assays 8 8 XylR wt XylR wt XylR1 XylR1 XylR2 XylR2 6 6 Promoter activity Pu (b-Gal, x 103) 4 4 2 2 0 0 10-4 10-3 10-2 10-1 1 10-4 10-3 10-2 10-1 1 3-MBA (mM) 3-NT (mM)

  22. 0 0.5 1 1.5 2 2.5 3 Inhibition by 3-NT 6 5 XylR wt 4 3 Promoter activity Pu (b-Gal, x 103) 3-MBA 1 mM 2 1 0 3-NT (mM)

  23. Structural prediction for the A domain of XylR Grupo de Diseño de Proteínas-CNB

  24. Contact surfaces protein/effector

  25. Mapping structural changes in the model F65L XylR2 46-50 XylR3 161-166 XylR5

  26. F65L XylR2 46-50 XylR3 161-166 XylR5 Mapping structural changes in the model

  27. F65L XylR2 46-50 XylR3 161-166 XylR5 Mapping structural changes in the model

  28. Mapping structural changes in the model L184I XylR4 V124A XylR1

  29. Mapping structural changes in the model L184I XylR4 V124A XylR1

  30. XylR wt XylR1 XylR2 XylR3 XylR4 XylR5

  31. Loops involved in the effector pocket

  32. Conclusions • Mutants XylR1 to XylR5 bear changes that unlock the ability of XylR to respond to many non-natural effectors • The changes involve not only the shape of the effector pocket, but also the structural transmission caused by inducer binding

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