BIO 315 Lecture Notes - Lecture 6: Auxotrophy, Plasmid, Pseudomonas
9 Jun 2018
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Lecture 6: Bacterial Genetic Analysis
Introduction:
• The study of bacterial genetics has come a long way
• Early researchers in the field focused on microbes of practical importance
• Researchers today continue this work but are also focused on a greater understanding of
the genetic potential of microbes
• This chapter explores the field of bacterial genetics, including potential application of the
information obtained
• Bacteria are used to remove toluene (pseudomonas)
Bacteria as Subjects of Genetic Research
• The birth of bacterial genetics
• Bacteria = ideal candidates for genetic research
o One chromosome = easy detection of mutations
• In early studies, prototroph (parental strain/ wild type) and nutritional mutants (auxotroph)
were used
o Auxotroph have additional requirements
o Allowed study of one gene based on its inability to utilize (or produce) a particular
environment
o Lederberg's experiment
▪ Reversion: Met- bacteria being cultured on Met- dish; some grow because
mutation occurred
▪ Genetic information is shared by bacteria
• Colony: visible to the naked eye; at least a million bacteria present
• Strain: cells in a colony that have different genetics than the other ones that came from a
single mother cell
• Organization of bacterial genomes
• Single chromosome and plasmid (if any)
o Termed the "replicon"
o Plasmid copy number in the cell is closely regulated
• Bacteriophage DNA may also be present
• Plasmids can't use identical copy number control mechanisms in the same cell (plasmid
incompatibility)
• One plasmid "loses out" not being replicated
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Mutations, Mutants, and Strains
• Mutants and mutations
• Microbial geneticists work to compare wild-type strains and mutant strains of bacteria
o The goal is to identify differing alleles of genes
• The genotype of an organism describes its collection of alleles of a given set of genes
o General designation "rules":
▪ A gene is given a three-letter abbreviation in italics, followed by a capital letter
to separate genes in the same pathway (ex: lacY)
▪ A protein is given the same three-letter designation but with the first letter
capitalized and no italics (ex: LacZ)
• Changes in genes are often visible by changes in phenotype or growth patterns
• Mutations can be made from UV light and chemicals
• Detection of mutants
• Phenotypic selection
o CFU = colony forming unit
o Use of a growth medium that will inhibit microbes lacking the desired genes
o Antibiotic selection is commonly used
▪ Culture sensitive to streptomycin are cultured on a medium containing
streptomycin
▪ Many died and after incubation mutations occurred and some became resistant
• Phenotypic screening
o Duplicate plates are created
▪ The first is under full nutritional support
▪ The second lacks a particular nutrient
o Where a olony grows on the fully supported plate, ut doesn’t grow on a partially
supported plate, a mutation has occurred
• Replica Plating
o Imprint colonies from master plate onto velvet
o Transfer colonies onto replica plates
o Incubate and compare growth on replica plates
• Evolution in an Test Tube
• Lenski's work in 1988 illustrated the evolution of E.coli in a period of 75 days, increasing in
fitness
• Procedure:
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