1. Inferring an Origin of Replication With Computational Methods Brian Smith

2. Overview  Introduction  Methods  Results  Future work

3.  Pseudomonas as a pathogen  A cryptic megaplasmid found in Psuedomonas syringae  Phenotypic costs associated with large scale HGT Introduction P. aeruginosa P. syringae pv. aesculi

4. The Problem  Conjugation of pMP into P. aeruginosa has failed.  In other Pseudomonads the pMP is transferred successfully at high numbers.  Several reasons why this might be including host range, or genes on the pMP that may illicit P. aeruginosa resistance

5.  The pMP is sequenced and the annotations are mostly ‘hypothetical protein’  Various methods for predicating bacterial origins of replication for chromosomes  I wanted to see if similar methods would work on this plasmid  GC skew  Repetitive Motifs Searching for the Origin

6.  Previously shown that a dramatic shift in GC content is associated with the chromosome origin and terminus (Lobry 1996). GC Skew E. coli

7.  I Used seqinR and an R script to calculate GC across the pMP GC Skew myseq <- read.fasta(file = "Desktop/pMP.fasta", as.string = FALSE, forceDNAtolower = TRUE, set.attributes = FALSE, seqonly = TRUE, strip.desc = TRUE)

8.  I Used seqinR and an R script to calculate GC across the pMP GC Skew myseq <- read.fasta(file = "Desktop/pMP.fasta", as.string = FALSE, forceDNAtolower = TRUE, set.attributes = FALSE, seqonly = TRUE, strip.desc = TRUE) Origin of Replication

9.  oriFinder  DnaA boxes vary in size  E.coli’s is (TTATCCACA)  Programs like this require that you know your motif  Built a custom python script from scratch Repetitive Motifs

10.  Input: fasta file  Output to Terminal:  Sequence selection  Min count  # of Motifs found  Top 10 common Motifs found  Output to file:  Dictionary of Dictionaries containing motif, count, and sequence position Repetitive Motifs

13.  Blast unknown protein sequence in this region involved in replication  Engineer smaller plasmids containing min. tools for replication using restriction enzymes  Attempt to conjugate new minimalist plasmids into P. aeruginosa Current/Future Goals

14. print “Thank you & Questions?”