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

6. 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

7. 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 typeMutant #1Mutant #2

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

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

10. 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

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

12. 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

13. 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 TraSH Methodology: Probe Production

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

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

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

17. Using pMAR 1xT7 1)Isolated PA14 transposants (very efficient) 2)Successful ARB PCR and Sequencing of transposants 3)Confirmed transposition into PA14 genomic DNA 4)Successful PCR after genomic digestion and adapter ligation 5)Successful T7 transcription of PCR products (see gel)

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

19. Protential Problems: 1)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.

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

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

22. 1)Isolated PA14 transposants (3x10 5 transposants/mating) 2)Successful ARB PCR and Sequencing of transposants 3)Confirmed transposition into PA14 genomic DNA 4) Amp s confirms that plasmid sequence did not recombine Using pMAR 2xT7

23. Future Work Optimize ARB PCR with new Mar2xT7 transposon Southern to confirm Mar2xT7 has not transposed more than once in each transposant 3)Confirm TraSH probes can be produced efficiently 4) Construct Library 5) Produce PA14 microarray

24. 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

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

26. 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.

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

28. RNAi clones that give Esp phenotype RNAi cloneGene identityRNAi phenotype 45H5 unknown (38 aa) Adult Unc 53B7 unknown Thin, Clr 53G9 mel-11(emb. elong) Myosin phosphatase regulatory subunit 38E2 unknown 59C10 unknown Pvul, Egl, Unc

29. 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

32. Longevity Assay

33. Genomic Structure of F13B10.1

34. SARM Long SARM Short

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

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

38. 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

39. 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 A.Perera L. Gendal J. Xe P. Juels C. Xi R. Elliot L. Li Ruvkun Lab Sylvia Lee