Transcription Activator-Like Effectors (TALEs) are a new class of DNA-binding proteins which selectively bind to double-stranded (ds)DNA with the 12th and 13th amino acids of each repeat, termed repeat variable diresidues (RVDs), controlling the nucleotide specificity. TALE proteins can therefore be designed using RVDs to select for any target DNA sequence. In this study, TALEs were engineered to recognize specific DNA sequences of the stx2 gene encoding for Shiga toxin present in E. coli O157. We investigated a new approach using a plant expression system to improve the production of TALEs that were previously expressed in Escherichia coli. A transient plant expression system using the pEAQ vector and Nicotiana Benthamiana was used to express the two custom-designed TALE proteins. Each TALE was subcloned into different pEAQ vectors and transformed into A. tumefaciens, which were agroinfiltrated into the plants to start expression. Leaves were then harvested and the two proteins were isolated using Nickel columns. The purified TALE proteins were characterized and then labeled using quantum dot to develop dsDNA detection arrays.

Expression of TALEs in plants and development of TALE arrays for diagnostic applications.

2019 Spring National Meeting

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