Development Of An Inducible Suicide Vector For The Control Of Agrobacterium Tumefaciens

Open Access
Author:
Hamaker, Nathaniel K
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wayne Roger Curtis, Thesis Supervisor
  • Wayne Roger Curtis, Honors Advisor
  • Phillip E Savage, Faculty Reader
Keywords:
  • suicide
  • agrobacterium
  • plant biotechnology
Abstract:
Plants can be genetically altered to produce proteins or metabolites of interest following transfection with bacterial transformation vectors, mainly Agrobacterium tumefaciens. Currently, there are limitations when attempting to run bioreactor-scale systems due to overgrowth of the bacterial vector in plant cell and tissue cultures. Initially, some degree of co-cultivation is necessary to deliver the transgenes, but the infection must be terminated at a point through the use of expensive antibiotics (e.g. moxalactam), inhibitory to scale-up. Incorporating such a purge into a bioreactor on a larger scale is financially impractical. The focus of this work was to develop cost-effective mechanisms for providing control over the genetic transformation process as applied to the use of bacterial vectors in plant tissue reactors. A synthetic plasmid, harboring an inducible kill switch was used to simulate the desired bacterial phenotype of a controllable strain of A. tumefaciens that has yet to be generated. Specifically, the construction of a lethal DNA construct harboring a toxic operon was pieced together from a variety of coordinated genetic elements sourced from E. coli and bacteriophage T7. Genetic transcriptional barriers were used to suppress basal levels of expression system to prevent death when undesirable. Endogenous Toxins are only produced when triggered by specific chemical signals, designed as a cost-effective alternative to the aforementioned antibiotics. The construct is intended for eventual insertion into the genome of the bacteria to create a stable strain with a sleeping kill switch. As transgenic proteins and other plant-derived products have a wide range of lucrative applications in the agriculture pharmaceutical industries, such a strain represents a highly desirable asset to any company involved in plant biotechnology.