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Cloning Project

Background
The avrPto gene in Pseudomonas syringae pv. tomato bacteria creates a protein that appears to play a role in resistance of certain host plants. Plants containing the Pto resistance gene produce a plant protein that interacts with the bacterial avrPto protein. To learn more about this interaction and the role these genes play in the defense response pathway I was able to play a small part in a mutagenesis study being conducted by Li Wong in Dr. Adam Bogdanove's lab at ISU
Prior to my arrival Dr. Bogdanove’s lab had created single nucleotide mutations in the avrPto gene using PCR. Li had 2 mutations of the gene for me to work with; G99V and S147R.

Objective
The goal of the research is to develop strains of Pseudomonas that contain these mutated forms of avrPto and determine the effect, if any, of the mutation on the plant-pathogen interactions.

Problem
We begin with G99V and S147R in plasmid pCPP2329PP, a plasmid that is not expressed well in Pseudomonas bacteria. Another plasmid, pML123, is better suited to express G99V and S147R in Pseudomonas, however specific restriction sites must be used to insert our mutated fragments into this vector. Unfortunately, the restriction sites we need to use are not found on the pCPP2329PP plasmid where we currently have the G99V and S147R fragments.

Solution
We hope to remove the G99V and S147R fragments from the current plasmid and insert them into an intermediate plasmid, pCR-Blunt-II-TOPO. The Blunt-II-TOPO plasmid contains corresponding restriction sites with the Pseudomonas specific vector pML123.

Vector map of Invitrogen pCR-Blunt-II-TOPO

Method

Cut the desired fragments from the current plasmid with restriction enzymes
Dephosphorylate the 5’ ends of the fragment with CIAP and insert the fragments into the Topo II plasmid
Use electroporation to transform the Topo II plasmids into competent E. coli cells
Isolate the Topo II plasmids from selected colonies and confirm the presence of the fragments with PCR
Cut the G99V and S147R fragments from the Topo II plasmids with restriction enzymes and separate using electrophoresis
Recover the fragments from the gel and use ligase to insert them into pML123
Transform pML123 vectors into competent E. coli cells with heat shock
Isolate the pML123 plasmids from selected colonies and confirm the presence of the G99V and S147R fragments with PCR
Use western blot to see if the avrPto protein is expressed in the pML123 when induced
Transform the pML123 plasmids into Pseudomonas

Results
To date we have had success with G99V and currently we have ligated it into the pML123 plasmid. We are in the processes of inducing expression of the avrPto protein.

S147R has been more problematic. We have not been able to successfully ligate the S147 into the Topo II plasmid.