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Video aid: Plasmid Cloning + Not (1) EcoRV (3500) ori Kane Amp Kont (900) PGREEN 4000 bp GFP Pul (2200) Bg/II (1800) [1] 1. Explain the function of the promoter sequences (P) present on the plasmid. 2. Explain why ampicillin acts as an antibiotic, and the mechanism whereby the ampgene functions in bacteria. [2] 3. Which RE site on DGREEN will be the most suitable for cloning of your PCR amplicon? Motivate your answer. [2] 4. You prepare a linear AGREEN plasmid by digestion with the enzyme identified in (3) above, ligate it to the PCR product, and transform it into bacteria. What phenotype wold you expect for bacteria that have taken up recombinant plasmids, versus those with non-recombinant plasmids? [3] 5. With a non-directional doning such as this, recombinant plasmids can contain an insert ligated into the vector in two different orientations. Explain how a double digest with Noti and Rsal will allow you to distinguish between these two types of recombinants. [2] 6. Will a bacterium containing a recombinant plasmid be able to the express the part of the human protein encoded by the HFE gene exon 4? Motivate your answer [2]