Defining Stage-Specific and Constitutive Promoters in Plasmodium yoelii

Open Access
Author:
Finger, Logan
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Scott Lindner, Thesis Supervisor
  • Joseph Reese, Honors Advisor
Keywords:
  • Biochemistry and Molecular Biology
  • Malaria
  • Plasmodium
  • CRISPR/Cas9
  • Stage-Specific Promoters
  • Genetically Attenuated Parasites
Abstract:
Developmentally attenuated malaria parasites have shown to be of interest due to their potential to be live-attenuated vaccine candidates. An increasingly common way to modify parasites so that the effects of essential gene deletions/modifications can be studied is through the use of CRISPR/Cas9. In this series of experiments, I show through the use of fluorescence microscopy and flow cytometry that HSP70-2/BiP, which is active within all stages of the parasites life cycle, is capable of driving production of green fluorescence protein (GFP) constitutively in vivo. In tests comparing different lengths of the BiP promoter, I seek to determine the minimal composition of the promoter can be such that it is still functional. Likewise, these tests help establish what qualifies as the minimal promoter for HSP70-2/BiP. Determining this will help with plasmid construction by diversifying the promoter types used within transgene expression plasmids. By doing this, the number of recombination events that tend to occur during replication of the plasmid within bacteria or within the parasite after transfection can be minimized. Additionally, elucidating a correlation between promoter composition and expression level could be useful in controlling expression levels in vivo. Similarly, through testing different stage-specific promoters using fluorescence microscopy and immunofluorescence assays, I can determine when a given promoter is active throughout the lifecycle of the parasite. This information could be used to transcribe desired transgenes such as the Cas9, protein and sgRNAs to enable the targeting of essential genes at specific stages of the parasite’s lifecycle, thereby producing developmentally attenuated parasites capable of infecting a host but with no ability to mature further. If these developmentally arrested parasites were used in a vaccine, they could effectively infect a host but be unable to cause an infection. Thereby, this would expose the body to parasites against which antibodies and other immune responses could be produced but without the risk of developing the disease. In addition to CRISPR, other genetic tools such as CRE/lox recombination, DiCre recombination, and promoter swap experiments also serve to benefit from stage-specific promoters.