PA14 Unigene Library Construction and

Screen for Esp Phenotypes

Nicole T. LiberatiGroup 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 a) RNAi Screen b) Reverse Genetic Screen

of Candidate Esp Genes

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

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Num

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PAA.1(100 pl.)

PAA.2(200 pl.)

PAA.3(300 pl.)

PAA.4(400 pl.)

PAA.5(500 pl.)

PAA.6(600 pl.)

PAA.7(700 pl.)

PAA.FIN1(+89R)

PAA.FIN2(+785R)

Assembly Name

Contig Number/Read Number Analysis

Contigs >5 ReadsContigs >10 ReadsContigs >20 ReadsContigs >50 Reads

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

Unigene Library:Unigene Library: A collection of A collection of P. aeruginosaP. aeruginosa strains strains containing a disruption in each containing a disruption in each non-essential open reading frame (ORF) non-essential open reading frame (ORF) in the in the P. aeruginosaP. aeruginosa genome genome

Wild type Mutant #1 Mutant #2

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

Genomic DNA

Transposon

Transposon-specific Primer

Arbitrary PCR Primers

LEGEND

1st PCR Reaction

2nd PCR Reaction

PCR Cleanup and Sequencing

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2

2

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Disrupted Gene Identification

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

TnPhoA

Neo resistance

Transposase

Amp resistance

pir-dependent ori

Inserted into the PA14 genome:

p733

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

5) Reverse Transcription with fluorophore to produce labeled cDNA6) 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

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

pMFLGM.GB-T7 (pMAR 1xT7)

Gm resistance

Mariner Transposase

Amp resistance

ori R6K (pir+)

Inserted into the PA14 genome:

Tra genes

lacZ

Frt

Frt

109 bp IR

109 bp IR

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

Frt

109 bp IR

109 bp IR

Using pMAR 1xT7

1) Isolated PA14 transposants (very efficient)2) Successful ARB PCR and Sequencing of transposants3) Confirmed transposition into PA14 genomic DNA4) Successful PCR after genomic digestion and adapter ligation5) Successful T7 transcription of PCR products (see gel)

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

Frt

109 bp IR

109 bp IR

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.

pMycoMar

Neo resistance

T7 Promoter

T7 Promoter

29 bp IR

29 bp IR

ori R6K (pir+)

C9 Himar1 transposase

Gm resistance

cos site

Myco promoter

pMycoMar

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

29 bp IR

1) Isolated PA14 transposants (3x105 transposants/mating)2) Successful ARB PCR and Sequencing of transposants3) Confirmed transposition into PA14 genomic DNA4) Amps confirms that plasmid sequence did not recombine

Using pMAR 2xT7

Future Work

• Optimize ARB PCR with new Mar2xT7 transposon• Southern to confirm Mar2xT7 has not transposed more

than once in each transposant3) Confirm TraSH probes can be produced efficiently4) Construct Library5) Produce PA14 microarray

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

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

RNAi Screen - High Throughput Protocol

Dry RNAi O/N cultures onto RNAi platesIncubate plates O/N at RT

Add L1-stage N2 (daf2) worms to RNAi platesIncubate 48 hours at 20C - L4 Stage

Dry PA14 O/N culture on the RNAi platesIncubate at 25C.

Begin counting after 24 (40) hours at 25C.

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daf2-L4440daf2-pmk1wt-L4440wt-pmk1

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Utility of daf-2 resistance for RNAi library screening

Larger windowto work for +/-pmk-1 RNAi.

RNAi clones that give Esp phenotype

RNAi clone Gene identity RNAi 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

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

RNAi esp Candidates

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L4440RNAi-177RNAi-1RNAi-2RNAi-3RNAi-4RNAi-5RNAi-6RNAi-7RNAi-8RNAi-9RNAi-10RNAi-11RNAi-12

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Vector RNAiF13B10.1 RNAipmk RNAi

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Vector RNAiF13B10.1 RNAipmk RNAiOP50

Longevity Assay

Genomic Structure of F13B10.1

SARM Long

SARM Short

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

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N2-Vector RNAisek1-Vector RNAiN2-F13B10.1 RNAisek1-F13B10.1 RNAi

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

Future Work

1) Activated p38 Immunoblot on F13B10.1 treated worms2) Test for susceptibility phenotype on E. faecalis3) Isolate F13B10.1 null mutant

AcknowledgementsAusubel LabDan LeeJas VillanuevaSachiko MiyataJonathan UrbachTao Wei Dennis KimRhonda Feinbaum

Rahme LabJian Xin HeMaude Saucier

Rubin LabChris Sassetti

Mekalanos Lab

Partners Genome CenterR. KucherlapatiK. MontgomeryK. OlsonW. Brown J. DeckerA. PereraL. GendalJ. XeP. JuelsC. XiR. ElliotL. Li

Ruvkun LabSylvia Lee