Transcript If we should succeed in helping ourselves through applied genetics

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 (2n)
Start
Heat Denaturation (95 C)
Annealing (50-55 C)
(primer binding)
Elongation (polymerization)
(72 C)
Transformation
PCR
OR
Cloning
cDNA
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