Question

NOTE: this will require that you remember some stuff from bacterial genetics.

You want to research the eukaryotic protein BabyFeet, which smells like peppermint. To make cDNA you need...

a) You prepare plates to grow your bacteria on. Your competent E. coli are resistant to penicillin, cephalexin, and susceptible to ampicillin. Which antibiotic will you put in your media?

b) Next, you prepare your cloning vector, which contains the genes Ampr, cepr, and penr all downstream of the promoter. The plasmid map below shows you the locations of many restriction sites, indicated by the restriction enzyme (x______) that cuts at that spot. Assume these restriction enzymes will all work for your cDNA as well. Which restriction enzyme would be best for your experiment? Why?

xCant xYou Amp' - Sit promoter cep xWith Us pen xWith Us xMathletes

c) Describe how you will get your donor into your plasmid.

d) You isolate whole plasmids by gel electrophoresis. Then you transform your E. coli with the plasmid. Finally you plate E. coli onto the media you prepared with ampicillin. How will you know if your vector was successfully incorporated into the genome?

Answer 1

A)medium +penicillin+ cephelexin

B) vector figure is not shown

C) by using same restrcition enzyme digestion for both vector and genenand then ligation using enzyme DNA ligase.

D)after successful transformation ,E.coli can grow in penicillin+ cephelexin + ampicillin as plasmid having ampicillin as antibiotic marker.

Plasmid does not incorporate into genome of bacteria and if it incorporates into genome then whole gonome can be isolated and run on gel it's size will be increased due to insertion of plasmid.

Hope it's clear..thanks