The Precise Location of DJ1C in Plant Chloroplast

Undergraduate #35
Discipline: Biological Sciences
Subcategory: Plant Research

Alexandria Lauray - Clark Atlanta University
Co-Author(s): Rebecca Roston and Julie Stone, University of Nebraska-Lincoln

DJ1C is a member of a family of proteins found throughout life, including humans and plants, whose study in plants could potentially lead to a revolutionary cure for cancer and Parkinson’s disease. DJ1C has been found to be located in the chloroplast and in order to understand the function of DJ1C in chloroplast; we have to determine where it resides. This experiment is a two part process. The first part of the experiment is to ensure that we have properly placed our DJ1C insert into a vector in the proper direction. To isolate BRDJ1C and locate its precise location in chloroplasts, we performed a series of experiments to ensure the gene (with its chloroplast transit peptide, cTP) was inserted under control of the SP6 promoter in our vector. We first designed oligonucleotide primers engineered with restriction enzyme sites, a Shine-Delgarno sequence and a polyA tail , performed a PCR reaction to amplify our gene of interest, and then we began to clean and concentrate the DNA. After concentration, we performed gel purification, then quantified our sample using a Nanodrop and performed a ligation with the pGEMT-Easy vector. After transforming, we picked and cultured colonies to prepare for the next step of plasmid DNA preparation. Afterwards, we sent our samples off to sequencing and performed restriction enzyme digestions using EcoR1 to ensure our insert is incorporated in the correct orientation of the vector. The second part of the experiment is to import our DJ1C into the chloroplast with a fluorescently-labeled protein. To achieve this, we will use a coupled in vitro transcription/translation system and incorporate fluorescently-tagged lysine into the protein. Then, we will perform a chloroplast fractionation procedure to separate the soluble and insoluble regions of the chloroplast. After we separate the compartments of chloroplast, we will run it on a protein gel and detect the protein in question, DJ1C, in either the stroma or the thylakoid space. In further work we will continue to produce BrDJ1C protein driven with SP6 promoter using an in vitro transcription/translation system with fluorescently-labeled lysine, we will then import BrDJ1C into the chloroplast, and fractionate chloroplasts to isolate soluble (stroma) and insoluble (thylakoid) regions. Once we have done this, we will detect the DJ1C protein and therefore determine the location of DJ1C. Locating DJ1C will further our under-standing of its role in chloroplast development, and may lead to insights in the function of homologs in other organisms. Special acknowledgments go to The Roston Lab, Redox Biology Program at UNL and a Special thanks goes to Ms. Sharon Lauray, M.P.A, M.S.M.

References: ‘Chloroplast Diagram – Pictures, Photos & Images of Plants – Science for Kids.’ Chloroplast Diagram – Pictures, Photos & Images of Plants – Science for Kids. Web. 11 Aug. 2015. Lin et 2011

Funder Acknowledgement(s): NSF grant #DBI-1461240

Faculty Advisor: Julie Stone,