Bio 219 Final Exam 100% Verified!

Purpose of this experiment - ANSWERto create a genomic library of A. fischeri to create a restriction map of the plasmid - containing lux operon Overview of experiment in order - ANSWER1. Isolate chDNA from A. fischeri 2. Analyze purity and determine concentration 3. Restriction digestion / Shotgun Cloning 4. Ligation of insert and vector 5. Transformation / Screening 6. Re-isolation (Plasmid mini-prep) 7. Restriction mapping Characteristics of A. fischeri - ANSWER- gram negative - rod-shaped - lives in symbiosis with marine organisms - chDNA contains the lux operon - lux operon encodes for proteins that code for bioluminescence Characteristics of E. coli - ANSWER- gram negative - rod-shaped - lives in lower gut of warm-blooded animals - prokaryotic model organism as host for working with recombinant DNA Gram negative bacteria - ANSWER- external lipopolysaccharide (LPS) layer - single layer of peptidoglycan - does not retain stain Lipopolysaccharide (LPS) layer - ANSWER- varies between bacteria - O-antigen stimulates immune response Operon - ANSWER- system in which contains genes that encode for specific function - clustered on DNA sequence - allow protein synthesis is controlled and coordinated with needs of cell chDNA - ANSWER- large circular DNA (bacteria) - contains the lux operon Restriction Digestion - ANSWER- cut DNA into smaller pieces - enzymatic reaction via restriction enzymes - cuts at specific locations on DNA P