Culminating Project Title
Construction of a Site-Specific Integrative Vector for Use in Genetic Engineering of Cronobacter sakazakii
Date of Award
Culminating Project Type
Biological Sciences - Cell and Molecular: M.S.
College of Science and Engineering
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Keywords and Subject Headings
Cronobacter sakazakii genetic modification
Cronobacter sakazakii is a gram-negative bacillus belonging to the family Enterobacteriaceae and is classed as an opportunistic pathogen. Capable of mortality rates in excess of 40% in infected infants and neonates, many investigations have sought to elucidate the pathogenesis of this organism. Constructing a site-specific integrative vector that could be utilized to engineer the species and create a fluorescent C. sakazakii strain may provide a useful visual aid in tissue invasion studies. The vector pOSIP-KC was utilized as a plasmid backbone into which the φES15 integrase gene, derived from a C. sakazakii lysogenic phage, along with the φES15 attP attachment sequence were cloned, creating the novel integrative vector pOSIP-ES15. The efficiency of vector integration and targeting at the predicted attB location was tested by transforming electrocompetent C. sakazakii NCTC 11467 cells with the vector and confirming integration through PCR analysis. pOSIP-ES15 appeared to preferentially integrate into other unknown locations in the chromosome, only integrating into the attB site approximately one third of the time. An expression cassette with a gene encoding the Superfolder GFP protein, driven by the strong, constitutive promoter nptII, was cloned into the vector to create pOSIP-ES15_GFP. Through repeated attempts, although integration of the vector into the NCTC 11467 chromosome was verified, no visual fluorescence was detectable. Fluorescence analysis by spectrophotometry showed no significant difference in emission intensity between integrated strains and parental NCTC 11467 cells. Expression of the cassette when harbored in an episomal plasmid within NCTC 11467 led to high intensity fluorescence, indicating the strain could express the cassette, but once integrated into the chromosome unknown factors caused a silencing of expression. This project demonstrated that site-specific integrative vectors can be powerful genetic engineering tools, but that without prior characterization of integration locations, expression of exogenous DNA introduced cannot always be reliably predicted.
Ellis, Samuel D., "Construction of a Site-Specific Integrative Vector for Use in Genetic Engineering of Cronobacter sakazakii" (2020). Culminating Projects in Biology. 48.