BIO 172 1st Edition Lecture 17 Outline of Last Lecture I. Molecular BiologyOutline of Current Lecture I. Molecular BiologyII. MutationsCurrent LectureMolecular Biology:There are specific sequences that the promoter and gene share. The gene looks to bind at the specific matching sequence on the promoter. They Bind and then cut at THAT sequence. STICKY END: A promoter lets the gene come in and go from either end, because it has a Sticky End. Sticky is the same on both sides of the gene. BLUNT END: If you don’t have an overhang, it’s called Blunt. The gene may only attach to the promoter a specific way with the blunt end, because the match does not apply to both sides. So to create the correct protein, it must match the site to the promoter.DIRECTIONAL CLONING: needs two cut sites!When you want to clone the entire gene: Choose to use restriction enzyme sites that do not cut within the gene you’re cloning. You cannot use cut sequences that are located anywherewithin the coding region.The red arrows are enzymes youcannot use! Because they wouldcut somewhere within the gene.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.The 3’ end anneals, and through a PCR reaction, the 3’ end can be extended and then copy the restriction cut site. After a few completions, the product becomes the template. Then you have multiple copies of the single stranded template DNA. Cutting one of the restriction sites gives it the sticky ends. Question: Which restriction enzyme sites would you use and WHY to directionally clone a DNA fragment into this plasmid (allowing for transformation and propagation in bacteria cells)? Keep in mind, all enzymes listed generate sticky ends: Xho1, Bam HI, Amp, Nde 1, Ori, Eco RI. (origin is origin of replication)You choose the sites that remove an unimportant part of the gene!! Because then if the site removes part of the gene that is not important for function, then the protein can still work.Transformation: Genetic Transformation is alteration in a cell that results from uptake/expression of foreign genetic material.Laboratory technique for efficient transformations is to Increase Permeability of bacterial cell Membrane, by chemical treatment or electrical shock.Transformation=- uptake of some foreign type of DNA, so getting big molecules across the membrane. You can treat so that a membrane is more permeable so plasmids can get it. Shocking/heating them works for DNA to move across membrane.Western Blotting: detecting specific PROTEINS by a similar technique of using gel and transferring it to a membrane, the protein you look for will be obvious.Mutations:Mutations occur randomly, they are not selected for because of a change in the environment. Rather, if a mutation happens to be helpful in survival for some organisms, then that mutation might get passed on for a long