We have used E. coli as a chassis organism to allow us to clone our gene construct successfully. After using Golden Gate cloning to make our Level 1 and Level 2 constructs, we needed to transform E. coli with the newly made constructs. This is to ensure that we have enough of the correct construct when we get to the next stage of transforming Agrobacterium tumefaciens.
Agrobacterium tumefaciens
A. Tumefaciens (Agro) was a chassis organism in this project to act as a shuttle chassis for the delivery of our Level 1 and Level 2 DNA constructs into Nicotiana benthamiana. Agro infects the plant through its Ti plasmid. This inserts a segment of its DNA, T-DNA, into the host’s genome. This allows the expression of the gene of interest.
Nicotiana benthamiana
N. benthamiana (Benth) was selected as the plant host as it is a model organism for the plant world. Benth has been extensively studied for decades as has it’s interactions with Agro. It is also easily cultivated so there is no short supply in the laboratory.
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