What is a plasmid? A plasmid is an extra-chromosomal element, often a circular DNA. The plasmids we will use in this class typically have three important elements: Coiling in a plasmid You probably remember that double-stranded DNA has the form of a 'double helix' which looks a bit like a telephone handset cord (except that the telephone cord is a single helix). You may also recall that the double helix is right-handed (for an expose on the difference, take a look at the Left Handed DNA Hall of Fame Site.) You've probably also noticed how knotted up a telephone cord can get, if your roommate twists the handset around a few times before hanging up. Those knots are a higher order structure that lead to 'coiled coils.' DNA has the same problem, though your roommate isn't to blame this time! Aside from the double-helical structure that we all know and love, DNA can take on a higher order coiling that twists one double helix around another. We call this 'superhelical coiling' or simply 'supercoiling.' In a linear molecule these twists can unravel by themselves, provided the ends are not prevented from rotating. In a circular molecule with no free ends, the superhelical twists are 'locked in' and the molecule cannot relax. This coiling is not the same as the right-handed double helix coil with which you are all familiar. The supercoiled molecule is a coiled coil. You can click on the image below to see an electron micrograph of a supercoiled circular DNA. Relaxation What's needed to get supercoiled circular DNA to relax? A few weeks of pampering at a spa perhaps? No! If one of the two strands is broken so that it has free 5' and 3' ends, the supercoils can relax even though the overall structure of the molecule remains a circle. The free ends of the broken strand rotate around the phosphate backbone of the intact strand (the one that wasn't broken). This loss of superhelical stress puts the plasmid into a 'relaxed DNA' form. Another electron micrograph: This one is of relaxed DNA What is a vector? Plasmids are sometimes called 'vectors', because they can take DNA from one organism to the next. Not all vectors are plasmids, however. We commonly use engineered viruses, for example bacteriophage lambda, which can carry large pieces of foreign DNA. How do we isolate a plasmid we want? We introduce the reclosed (ligated) products into E. coli, a process called 'transformation', and select for bacteria resistant to a drug (such as amipicillin, for example). We screen the individual bacterial colonies to find one that contains a plasmid of the correct structure. When we are transforming DNA in the laboratory (i.e. for experimental purposes), we have several ways of making the uptake of DNA by E. coli cells more efficient. Source.


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Last Modified: April 18, 2016 @ 4:08 pm