Abstract
With the advances in the field of synthetic biology, there is an increasing demand for multi-gene cloning technologies. Molecular cloning to generate multi-gene constructs can be performed by restriction digestion, or by recombination-based cloning strategies such as Gateway®. This chapter details cloning, transformation, and selection procedures involved in generation of multi-gene expressing transgenic plants. Methods are described for cloning five distinct promoter–reporter fusion constructs into the PC-GW-BAR vector (from the PC-GW vector series) using Gateway® technology and meganuclease sites. Further, transformation and selection methods are described for the biofuel crop Camelina sativa from the Brassicaceae family. These methods would be constructive toward generating multi-gene expressing plants for simultaneous expression analysis of five promoters in a short time period.
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Acknowledgements
This work was conducted at departments of Crop Science and Plant & Microbial Biology at North Carolina State University, and was supported by DOE ARPA-E grant DE-AR0000207. The author thanks Dr. Ron Qu and Dr. Roopa Yalamanchili for review of the manuscript.
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Dalal, J. (2016). Simultaneous Analysis of Multiple Promoters: An Application of the PC-GW Binary Vector Series. In: Hehl, R. (eds) Plant Synthetic Promoters. Methods in Molecular Biology, vol 1482. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6396-6_13
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DOI: https://doi.org/10.1007/978-1-4939-6396-6_13
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