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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
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
Contig Number/Read Number Analysis
Number of Contigs
1800
1600
Contigs >5 Reads
1400
Contigs >10 Reads
1200
Contigs >20 Reads
Contigs >50 Reads
1000
800
600
400
200
0
PAA.1
(100 pl.)
PAA.2
(200 pl.)
PAA.3
(300 pl.)
PAA.4
(400 pl.)
PAA.5
(500 pl.)
PAA.6
(600 pl.)
Assembly Name
PAA.7
(700 pl.)
PAA.FIN1 PAA.FIN2
(+89R)
(+785R)
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:
Wild type
A collection of P. aeruginosa strains
containing a disruption in each
non-essential open reading frame (ORF)
in the P. aeruginosa genome
Mutant #1
Mutant #2
Selection of Unigene Library Mutants
30,400 insertions
Approximately 5 hits per ORF:
~6 Mb
Choose the most 5’ disruption
within the actual coding sequence
~4800 catalogued Unigene mutants
Disrupted Gene Identification
3
1
2
LEGEND
Genomic DNA
1
2
1st PCR Reaction
Transposon
Transposon-specific Primer
2nd PCR Reaction
PCR Cleanup and Sequencing
Arbitrary PCR Primers
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
Transposase
p733
Neo resistance
pir-dependent ori
Amp
resistance
Inserted into the PA14 genome:
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
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
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
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:
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:
Using pMAR 1xT7
1)
2)
3)
4)
5)
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)
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:
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
Myco promoter
C9 Himar1 transposase
29 bp IR
T7 Promoter
L cos site
pMycoMar
ori R6K (pir+)
Neo resistance
T7 Promoter
29 bp IR
Gm resistance
pMFLGM.GB- 2xT7 (pMAR 2XT7)
lacZ
Mariner Transposase
pir-dependent ori
29 bp IR
T7 Promoter
pMAR 2xT7
Gm resistance
T7 Promoter
29 bp IR
Tra genes
Amp
resistance
Inserted into the PA14 genome:
Using pMAR 2xT7
1)
2)
3)
4)
Isolated PA14 transposants (3x105 transposants/mating)
Successful ARB PCR and Sequencing of transposants
Confirmed transposition into PA14 genomic DNA
Amps confirms that plasmid sequence did not recombine
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
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
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
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.
Utility of daf-2 resistance for RNAi library screening
Fraction alive
1
daf2-L4440
daf2-pmk1
wt-L4440
wt-pmk1
0.5
Larger window
to work for +/pmk-1 RNAi.
0
0
24
48
72
time (h)
96
120
144
RNAi clones that give Esp
phenotype
RNAi clone
Gene identity
45H5
unknown (38 aa)
RNAi
phenotype
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
120.00
L4440
RNAi-177
Percent Alive
100.00
RNAi-1
RNAi-2
RNAi-3
80.00
RNAi-4
RNAi-5
RNAi-6
60.00
RNAi-7
RNAi-8
40.00
RNAi-9
RNAi-10
RNAi-11
20.00
RNAi-12
0.00
0
10
20
30
40
Hours
50
60
70
80
120.00
Vector RNAi
F13B10.1 RNAi
100.00
Percent Alive
pmk RNAi
80.00
60.00
40.00
20.00
0.00
0
20
40
60
80
Hours
100
120
140
Longevity Assay
120.00
Percent Alive
100.00
80.00
Vector RNAi
F13B10.1 RNAi
60.00
pmk RNAi
OP50
40.00
20.00
0.00
0
100
200
Hours
300
400
Genomic Structure of F13B10.1
SARM Long
SARM Short
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
Are F13B10.1 and sek-1 in the same pathway?
120.00
N2-Vector RNAi
sek1-Vector RNAi
Percent Alive
100.00
N2-F13B10.1 RNAi
sek1-F13B10.1 RNAi
80.00
60.00
40.00
20.00
0.00
0
10
20
30
Hours
40
50
60
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
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
Acknowledgements
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