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PA14 Unigene Library Construction and Screen for Esp Phenotypes Nicole T. Liberati

PA14 Unigene Library Construction and Screen for Esp Phenotypes Nicole T. Liberati Group Meeting 12/3/02. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction II. Esp Screen

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PA14 Unigene Library Construction and Screen for Esp Phenotypes Nicole T. Liberati

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  1. PA14 Unigene Library Construction and Screen for Esp Phenotypes Nicole T. Liberati Group Meeting 12/3/02

  2. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction

  3. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction II. Esp Screen a) RNAi Screen b) Reverse Genetic Screen of Candidate Esp Genes

  4. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction II. Esp Screen a) RNAi Screen b) Reverse Genetic Screen of Candidate Esp Genes

  5. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction II. Esp Screen a) RNAi Screen b) Reverse Genetic Screen of Candidate Esp Genes

  6. Unigene Library: A collection of P. aeruginosa strains containing a disruption in each non-essential open reading frame (ORF) in the P. aeruginosa genome Wild type Mutant #1 Mutant #2

  7. ~6 Mb Selection of Unigene Library Mutants 30,400 insertions Approximately 5 hits per ORF: Choose the most 5’ disruption within the actual coding sequence ~4800 catalogued Unigene mutants

  8. LEGEND Genomic DNA Transposon Transposon-specific Primer Arbitrary PCR Primers Disrupted Gene Identification 3 2 1 1 2 1st PCR Reaction 2nd PCR Reaction PCR Cleanup and Sequencing

  9. Current Status of the Unigene Library • 48 x 96 (4608) mutants created. • 14 x 96 (1344) sequenced. • Insertion site identification protocol optimized. • Accompanying database operational. • Public website available to search/request mutants: http://pga.mgh.harvard.edu/Parabiosys/resources/bacterial_mutants.php

  10. TnPhoA Transposase p733 Neo resistance pir-dependent ori Amp resistance Inserted into the PA14 genome:

  11. TraSH: Transposon Site Hybridization Allows end-user to monitor the presence/absence of transposon mutants Involves the hybridization of of genomic sequences adjacent to the transposon to similar sequences on a microarray

  12. TraSH Methodology: Probe Production 5) Reverse Transcription with fluorophore to produce labeled cDNA 6) Hybridize differentially labeled probe pools to microarray Sassetti, C. M., Boyd, D. H., and Rubin, E. J., (2001). PNAS 9812712-12717

  13. TraSH Methodology: Detection of the Presence or Absence of Tn Mutants in a Pool of Mutants Sassetti, C. M., Boyd, D. H., and Rubin, E. J., (2001). PNAS 9812712-12717

  14. pMFLGM.GB-T7 (pMAR 1xT7) lacZ Mariner Transposase ori R6K (pir+) 109 bp IR Frt Tra genes Gm resistance Frt 109 bp IR Amp resistance Inserted into the PA14 genome:

  15. pMFLGM.GB-T7 (pMAR 1xT7) lacZ Mariner Transposase ori R6K (pir+) 109 bp IR Frt T7 Promoter pMAR 1xT7 Tra genes Gm resistance Frt 109 bp IR Amp resistance Inserted into the PA14 genome:

  16. Using pMAR 1xT7 Isolated PA14 transposants (very efficient) Successful ARB PCR and Sequencing of transposants Confirmed transposition into PA14 genomic DNA Successful PCR after genomic digestion and adapter ligation Successful T7 transcription of PCR products (see gel)

  17. pMFLGM.GB-T7 (pMAR 1xT7) lacZ Mariner Transposase ori R6K (pir+) 109 bp IR Frt T7 Promoter pMAR 1xT7 Tra genes Gm resistance Frt 109 bp IR Amp resistance Inserted into the PA14 genome:

  18. Protential Problems: Due to length of IRs, every cDNA probe will contain approximately 120 bp of transposon sequence. 2) Hybridization of cDNA probes with similar transposon sequence has not been demonstrated.

  19. pMycoMar C9 Himar1 transposase Myco promoter 29 bp IR T7 Promoter ori R6K (pir+) pMycoMar L cos site Neo resistance T7 Promoter 29 bp IR Gm resistance

  20. pMFLGM.GB- 2xT7 (pMAR 2XT7) lacZ Mariner Transposase pir-dependent ori 29 bp IR T7 Promoter pMAR 2xT7 Gm resistance T7 Promoter Tra genes 29 bp IR Amp resistance Inserted into the PA14 genome:

  21. Using pMAR 2xT7 Isolated PA14 transposants (3x105 transposants/mating) Successful ARB PCR and Sequencing of transposants Confirmed transposition into PA14 genomic DNA 4) Amps confirms that plasmid sequence did not recombine

  22. Future Work • Optimize ARB PCR with new Mar2xT7 transposon • Southern to confirm Mar2xT7 has not transposed more than once in each transposant • Confirm TraSH probes can be produced efficiently 4) Construct Library 5) Produce PA14 microarray

  23. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction II. Esp Screen a) RNAi Screen b) Reverse Genetic Screen of Candidate Esp Genes

  24. Requirement for a p38 MAP kinase signaling pathway in C. elegans immunity PATHOGEN ??? ESP-8 (MAPKKK) ESP-2 (MAPKK) PMK-1 (p38 MAPK) ??? ??? IMMUNE RESPONSE

  25. RNAi Screen - High Throughput Protocol Dry RNAi O/N cultures onto RNAi plates Incubate plates O/N at RT Add L1-stage N2 (daf2) worms to RNAi plates Incubate 48 hours at 20C - L4 Stage Dry PA14 O/N culture on the RNAi plates Incubate at 25C. Begin counting after 24 (40) hours at 25C.

  26. Utility of daf-2 resistance for RNAi library screening Larger window to work for +/- pmk-1 RNAi.

  27. RNAi clones that give Esp phenotype

  28. I. PA14 Unigene Library a) PA14 Genomic Sequence b) Library Construction II. Esp Screen a) RNAi Screen b) Reverse Genetic Screen of Candidate Esp Genes

  29. Longevity Assay

  30. Genomic Structure of F13B10.1

  31. SARM Long SARM Short

  32. Requirement for a p38 MAP kinase signaling pathway in C. elegans immunity PATHOGEN ??? ESP-8 (MAPKKK) ESP-2 (MAPKK) PMK-1 (p38 MAPK) ??? ??? IMMUNE RESPONSE

  33. Are F13B10.1 and sek-1 in the same pathway?

  34. Future Work 1) Activated p38 Immunoblot on F13B10.1 treated worms 2) Test for susceptibility phenotype on E. faecalis 3) Isolate F13B10.1 null mutant

  35. Acknowledgements Ausubel Lab Dan Lee Jas Villanueva Sachiko Miyata Jonathan Urbach Tao Wei Dennis Kim Rhonda Feinbaum Rahme Lab Jian Xin He Maude Saucier Rubin Lab Chris Sassetti Mekalanos Lab Partners Genome Center R. Kucherlapati K. Montgomery K. Olson W. Brown J. Decker Perera L. Gendal J. Xe P. Juels C. Xi R. Elliot L. Li Ruvkun Lab Sylvia Lee

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