Progress 04/01/13 to 09/30/14
Outputs Target Audience:
Nothing Reported
Changes/Problems: The experimental approach did not yield the desired mutants. What opportunities for training and professional development has the project provided? Two undergraduate students were employed in this project. The project did give them an experience with basic research and as a result, one of the students has settled in on some career goals. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? This is the project final report.
Impacts What was accomplished under these goals?
The goal of this project was to create mutants in Salmonella enterica serovar Typhimurium that were phase locked in either the virulent or avirulent phenotype using directed transposon mutagenesis. The approach remains plausible but in the time frame of the project the desired mutants were not selected.
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Progress 04/01/13 to 09/30/13
Outputs Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project started employing the research technician in the laboratory. However, shortly after the initiation of the project she resigned to pursue a Ph.D. degree in Ireland. Currently, two undergraduates have been employed to continue this work. The project has given the two students new opportunities for hands on research including media preparation, gene cloning, transposon mutagenesis, and bacteriophage mediated transduction. The students are in the process of learning these new skills. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Once the omp::phoA fusion is verified both fusions (sipB::lacZ and omp::phoA) will be moved into the virulent phenotype of S. enterica strain 798 by P22 transduction. This new strain will be mutagenized using a transposon and mutants that knockout the virulence phenotype identified. The location of the transposon insertion will be identified by DNA sequencing. In the second specific aim, we will move the mutation caused by the transposon (or use more classical mutagenesis techniques) to knockout the virulent or non-virulent phenotypes. These mutants will represent locked on or locked off mutants and they will be tested for virulence first in mice and then in pigs.
Impacts What was accomplished under these goals?
The work performed was specific to the first specific aim of the project: the selection of phase locked off mutants. To date we have cloned the two indicator genes from Salmonella enterica (sipB and ompF) individually into the cloning vector pBR322. These constructs were verified by PCR amplification followed by electrophoretic separation and by DNA sequencing. The second step was to place unique fusions at the 3’prime ends of the two cloned genes (lacZ for sipB and phoA for ompF). The sipB::lacZ fusion has been constructed and verified by PCR. Potential ompF::phoA fusions have been made and these are currently being screened to identify the correct fusions. Once the omp::phoA fusion is verified both fusions will be moved into The virulent phenotype of S. enterica strain 798 by P22 transduction. We have prepared the P22 lysates, tittered them, and this reagent is ready for this step.
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