Faculty Mentor
C. Neal Stewart, Jr.
Department (e.g. History, Chemistry, Finance, etc.)
Plant Science
College (e.g. College of Engineering, College of Arts & Sciences, Haslam College of Business, etc.)
College of Agricultural Sciences & Natural Resources
Year
2018
Abstract
Chloroplast genetic engineering is unique in that it allows for high levels of gene expression while providing a means of natural bio-confinement. Although the chloroplast genome sequence of over 800 plant species is known, the elements that naturally regulate chloroplast gene expression are poorly understood. Recently, our lab has developed a 264 part modular cloning kit that contains known chloroplast regulatory elements. This kit can be used for the construction of novel chloroplast transformation cassettes; however, functional testing of these cassettes is currently hindered by standard chloroplast transformation technologies. Therefore, the aim of this experiment was to develop a rapid cell-based screening method that can be used for analyzing chloroplast transformation vectors. Golden Gate cloning was used to assemble a cassette from our kit that contained the native chloroplast Prrn promoter driving expression of a spectinomycin resistance/green fluorescent protein gene fusion. The cassette was introduced into potato (Solanum tuberosum) suspension cells via particle bombardment and the cells were screened as early as 48 hours for GFP expression. The results of this study could significantly enhance chloroplast genetic engineering efforts by allowing for rapid testing of chloroplast regulatory elements and by accelerating the screening time of novel chloroplast transformation vectors.
Included in
Agricultural Science Commons, Biotechnology Commons, Plant Breeding and Genetics Commons
The use of potato (Solanum tuberosum) suspension cells for rapid screening of chloroplast transformation vectors
Chloroplast genetic engineering is unique in that it allows for high levels of gene expression while providing a means of natural bio-confinement. Although the chloroplast genome sequence of over 800 plant species is known, the elements that naturally regulate chloroplast gene expression are poorly understood. Recently, our lab has developed a 264 part modular cloning kit that contains known chloroplast regulatory elements. This kit can be used for the construction of novel chloroplast transformation cassettes; however, functional testing of these cassettes is currently hindered by standard chloroplast transformation technologies. Therefore, the aim of this experiment was to develop a rapid cell-based screening method that can be used for analyzing chloroplast transformation vectors. Golden Gate cloning was used to assemble a cassette from our kit that contained the native chloroplast Prrn promoter driving expression of a spectinomycin resistance/green fluorescent protein gene fusion. The cassette was introduced into potato (Solanum tuberosum) suspension cells via particle bombardment and the cells were screened as early as 48 hours for GFP expression. The results of this study could significantly enhance chloroplast genetic engineering efforts by allowing for rapid testing of chloroplast regulatory elements and by accelerating the screening time of novel chloroplast transformation vectors.