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2012 - 11th Annual NC State Summer Undergraduate Research Symposium
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Session Time :
8/1/12 3:00 PM - 8/1/12 4:14 PM
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Content Area : OIA-SRE, Office of International Affairs - Summer Research Experience
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Lead Student Presenters : Zhaoyue Shi
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Abstract Title : A Recombineering Gene System in Arabidopsis
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Abstract :
The goal of my project is to generate whole-gene GFP fusions by means of recombineering. The recombineering technology utilizes a homologous recombination system engineered in bacteria to manipulate any DNA sequence carried in the bacteria.
This methodology has several advantages when compared with other classical approaches. For example, the same experimental conditions can be used for any gene independently of its size. It also allows for the manipulation of large DNA fragments ensuring the presence of all regulatory sequences of the gene of interest.
Specifically, my project consists of tagging with a reporter a set of Arabidopsis genes related to the biosynthesis and transport of the plant hormone auxin. The genes contained in large transformation-competent bacterial artificial chromosomes (TACs) are first introduced by electroporation into the recombineering E. coli strain SW105. Existing recombineering cassettes are amplified by PCR using primers containing the required 50 nt gene-specific sequences necessary to target the cassette to the desired location in the gene of interest.These PCR fragments are then electroporated into E. coli and recombination is induced by heat shock to introduce the reporter gene into the desired location in the gene of interest.
Next, the selection marker present in the recombineering cassette is removed by means of an inducible flipase also engineered in the SW105 E. coli strain. Finally, upon sequence verification, the modified TAC clones are transferred to Agrobacterium. These Agrobacterium strains will then be used to transform plants and to study the expression patterns of the genes of interest.
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Mentor and/or Co-Author : Jose Miguel Alonso
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