Caroline Taylor
Year: 2016, 2017

“Functional Domain Analysis of the PSRP7 PETs Protein in Arabidopsis thaliana

Project Summary

Plastid-Specific Ribosomal Proteins (PSRP) are nucleus-encoded proteins which play a role in chloroplast translation, so named because the genes exist only in plants, having no orthologues in bacteria.  All PSRPs in Arabidopsis thaliana have been characterized except for PSRP7, in which a mutation is embryo lethal.  Plants with a mutation in the homologous gene of Setaria viridis die as seedlings due to a deficiency in chloroplast translation.  PSRP7 is encoded by the PETs gene (Polyprotein of Elongation-Translation factor-Ts), which contains two S1 domains and two EF-Ts (Elongation-Translation factor-Ts) domains. It was proposed that the protein translated from PETs must be cleaved to function, so isolating the domains of the protein and transferring them to mutants should show which domains play the necessary role to keep plants from dying.

The experimental plan is to perform PCR using primers with BP tags to amplify the DNA which codes for the domains of interest, purify these products, and use them for a BP reaction.  The BP reaction is the first step of a Gateway transformation, followed by an LR reaction to transfer the genes of interest from entry vector to destination vector.  Additionally, A. thaliana plants will be genotyped to identify individuals with one good copy of the PETs gene and one mutant copy, and a floral dip will be used to transfer the resulting products of the Gateway transformation to the plants.

My Experience:

Working in a lab at BTI for the summer has been one of the best experiences of my life.  I was part of the PGRP high school summer internship program for the summers of both 2016 and 2017.  My first summer, I was surprised to learn that I would be doing more than shadow my mentor, but complete and present my own research project.  I quickly became independent and did much of my project with little assistance.

The next summer was a different experience, but still very worthwhile.  I learned more lab procedures, but I also had a higher level of interaction with the other people in my lab.  Difficulties in the lab made me more dependent on my mentor’s guidance, but a closer relationship with her and the rest of the people in my lab made this experience even better. I felt much more connected to the other people I worked alongside, and got a greater feeling of what it would be like to work with people in a lab.