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KEY PLASMID DESIGN

Random Brick formation (Components of plasmid)

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These bricks are formed from a random soup of characterised promoters "P", a reporter gene fluorescent protein, and a random terminator "T". This uses BSA I sites already digested previously into the DNA in order to ligate randomly in the correct order to create a random, yet purposeful and functional fluorescent signal.

Brick stitching

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These bricks are then stitched together via amplifying each randomly assembled brick through common amplification sites and then cutting them using a set of restriction enzymes which give each plasmid a specific order of bricks, depending on which are cut and then ligated together. As shown.

Plasmid Design

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These methods are used to create two plasmids, both of which to some extent are randomly assorted. An sgRNA plasmid which complements the dcas9, and a reporter plasmid which expresses the randomly inhibited and produced reporter FP's along with a dcas9.