topic 9 Flashcards
history
- started research in 1940s
- studied e coli mainly
- studied to determine practical importance of microbes
- today study genetics to understand genetic potential of microbes
- were able to research by comparing to mutants
organization of bacterial genomes
- single chromosome
- plasmids
- bacteriophage dna also present
- anything being replicated is a “replicon”
plasmids
- not necessary for survival of bacteria, no “housekeeping” genes
- typically smaller than genomes
- however important because contain antibiotic resistance
- regulates its own replication in cell, may be more than one copy
mutations
bacteria are ideal genetic research candidate because
- one chromosome, so easy detection
- nutritional mutants were used, and studied ability to produce particular nutrient or not
eg. Chemotaxis
- capillary tube filled with nutrients
- microbes with normal chemotaxis will move into tube due to protein
- mutants without chemotactic protein remain outside
wild type
- strain like one found in nature
- original isolate
mutant
strain carrying mutation relative to wild type
mutation
change in gene that disrupts function
allele
variant of gene
- may gain function
- may lose function
- may change function
auxotroph
mutant is unable to make particular compound
- often cant make specific amino acid is requires
prototroph
strain capable of making all required organic compounds
nomenclature of genes
- three letter abbreviation in lowercase italics with capital letter for separ ate genes
nomenclature of proteins
- three letter abbreviation with first letter capitalized and capital letter to separate gene
genotype
- description of allele in an organism
- reflects differences of mutant from wild type
- eg. gene involved in histidine synthesis
phenotype
observable properties of a strain
- eg. strain unable to grow in absence of histidine
figure 9.1
- three cultures parental, mutant 1, mutant 2
- parental is a prototroph
- mutant 1 cant make methionine so no growth
- mutant 2 cant make proline so no growth
type of mutant
- change in genes
- visible by changes in phenotype or growth pattern
- -> physical, visual, observable change
selection mutation
- isolating cells of a particular genotype on growth basis
- have growth advantage if will kill wild type cells
screening mutation
- identification of cells with a phenotype
- colour, morphology, no growth
- no advantage or disadvantage
phenotypic selection
- use of growth medium that will inhibit microbes lacking desired genes
- antibiotic selection is commonly used
- eg kill wild type leaving for mutant
phenotypic screening
- duplicate plates with one lacking particular nutrient
- mutation is spotted when colony grows on full support plate, but not on partial support plate
- kill mutant, not wild type
replica plating
- create duplicate plates
- stamps growth mediums on velvet sheet and compare growth
patching
- transfer colonies to gridded plate
- more accurate and reproducible than standard velvet replica plating
- pick colony with sterile toothpick, sequentially inoculate gridded test plates with picked colony and incubate, and compare growth on test plates
types of mutation (3)
silent: no change in amino acid sequence
missense: change in codon that results in coding for different amino acid
nonsense: change forms a stop codon where one shouldnt be
frameshift: results in insertions or deletions of nucleotides that can alter amino acids sequences
reversion
a mutation that corrects a metabolic abnormality back to wild type form
- problematic when trying to determine mutation rates of chemical or dna exchange rates between microbes
- -> adaptive evolution (giraffe necks)
esther lederberg, when and how mutations occur?
- created by esther lederberg
- questioned when mutations arised
- imprint antibiotic plate, and transfered growth onto replica plate
- incubate replica plates
test for streptomycin resistance
–> since cells that werent exposed to antibiotic developed resistance, shows that mutations can arise from absence of selective agent
luria and delbruck also aid in determining when and how mutations occur
show variable resistance to phage infection arises in bacteria without selective pressure
- therefore concluded that natural selection also applies to bacteria
richard lenski
- illustrated evolution
- cultures with extended generational time without selective pressures had enhanced the ability to grow in culture compared to ones that were stored
comparison of ancestral culture and evolved culture
- mix cultures in one to one ratio
- shows that end up with evolved cultures through natural selection,
- relative fitness increases over time,
relative fitness
ratio of number of colonies
restriction enzymes (REs)
- produced by bacteria
- cut DNA at specific recognition site
- recognition sites are usually palindromic (reads forwards and backwards the same)
- similar ends of cut DNA can be paired together and ligated
- each base should come up 25% of the time in sequence
modification enzyme
- restriction enzymes are always paired with modification enzymes
- recognize the same site as paired restriction enzyme
- methyltransferase activity protects dna from endonuclease activity
dna ligase
the fragments are joined by dna ligase
cloning vectors
- REs allows researchers to stich together fragments of useful DNA into recombinant molecules
- recombinant molecules can be used to clone a bacterial gene of interest
- vectors are used to insert recombinant DNA molecules into a recipient host bacterial cell
- plasmids
- phages
- cosmids
plasmid cloning vectors
- cut fragments from two plasmids carrying anti biotic resistance genes with same RE
- transformed strain exhibited traits from both plasmids
necessary traits
- origin of replication
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