Chapter 16 Microbial Genomics “If we should succeed in helping ourselves through applied genetics before vengefully or accidentally exterminating ourselves, then there will have to be a new definition of evolution, one that recognizes a process no longer directed by blind selection but by choice.” Genetic engineering: the manipulation of DNA to introduce new genes to an organism or to modify existing genes --- transgenic, an organism carrying genes from a different individual --- can occur naturally among the bacteria --- generally requires human intervention among eukaryotes

Most DNA manipulation is done in bacteria Bacterial Advantages: 1.) rapid growth on simple substrates 2.) stable extrachromosomal DNA (plasmids) 3.) molecular tools, some model bacteria, particularly E. coli, are very well understood at the cellular level The most significant molecular tool was the discovery of restriction endonucleases. --- discovered in the early 1970’s --- cut dsDNA only at specific nucleotide sequences --- several hundred known --- it has recently become possible to design RE’s to cut at particular sequences --- are part of host restriction system, a way for bacteria to protect themselves from “foreign” DNA, relies on methylation to mark their own DNA

Restriction Endonuclases (enzymes) “Sticky” Ends Blunt Ends

Plasmid Cloning Cut (RE) Ligate Screen

Polymerase Chain Reaction (PCR) --- another way to get insert DNA for plasmid cloning --- basically PCR is DNA replication outside of a cell --- requires some knowledge of nucleotide sequence of the target DNA (primers) --- uses a thermo-stable DNA polymerase --- each cycle doubles the amount of target DNA (2 n ) Heat Denaturation (95 C) Elongation (polymerization) (72 C) Annealing (50-55 C) (primer binding) Start

Transformation Cloning PCR cDNA OR Cloning a Eukaryotic Gene --- ultimately want gene In an expression vector

Computer Based Sequence Annotation: --- pretty good, but not perfect E. Coli K-12 (MG1655): Genome size: 4.6 Mbp Predicted ORF’s (genes): 5,295 ( 88% of genome) Unknown function: 38%

DNA Microarray Data