Module 9 Flashcards
Microbial Genetics
1
Q
define: microbial genetics
A
study of DNA-encoded functions
2
Q
what forms can bacterial genomes take
A
- single or multiple chromosomes
- plasmids
- bacteriophage DNA
3
Q
what form do bacteria genomes usually take
A
one circular chromosome, possibly a plasmid as well
4
Q
how does the plasmid copy number vary
A
plasmid copy number is very carefully regulated
5
Q
define: prophage
A
bacteriophage DNA integrated within the genome of a bacterium
6
Q
define: replicon
A
all the chromosomes and plasmids that replicate within a bacterial cell and are passed on to subsequent generations
7
Q
how do plasmids replicate
A
self-replicating, replicated by host DNA polymerase
8
Q
what sorts of genes are encoded by plamids
A
those that are not essential housekeeping genes
e.g.
- enabling making nodules on leguminous plants
- introducing tumors
- ability to degrade certain compounds
- ability to confer metal resistance
- ability to be pathogenic
- antibiotic resistance
9
Q
what is the size of plasmids
A
smaller than most host genomes
10
Q
is losing a plasmid lethal
A
no, genes are non-essential
11
Q
define: wild type
A
- strain most like that found in nature
- source for deriving mutants
12
Q
define: mutant
A
strain derived from its wild type that carries a mutation
13
Q
define: allele
A
gene variant associated with the mutant strain
14
Q
what are the categories of alleles
A
- gain-of-function
- loss-of-function
- change-of-function
15
Q
define: auxotroph
A
strain that cannot make an organic compound required for growth
16
Q
what is the most common example of auxotrophic mutants
A
mutation in amino acid biosynthesis
17
Q
define: phototroph
A
nutritionally wild-type strain that does not need any additional growth supplement, often the parental strain that gave rise to the auxotrophic mutant
18
Q
how are genes named
A
three-letter abbreviation in italics, followed by a capital letter to separate genes in the same pathway
19
Q
how are proteins named
A
same three-letter abbreviation designation as genes but with the first letter capitalized and no italics
20
Q
define: genotype
A
description of alleles within an organism, generally reflects differences from wild-type
21
Q
define: phenotype
A
observable properties of a strain
22
Q
take the hisC gene, what does hisC- indicate
A
a mutation in the hisC gene, such that it cannot make its own histidine
23
Q
what is the phenotype of a mutant that cannot make histidine
A
His- mutant
24
Q
how does screening for mutants work
A
- separating & growing individual cells to look for a mutant phenotype
- grown on agar place or in liquid medium in multi-welled plates
25
when is screening for mutants used
when there is no growth advantage, nonselectable mutations
26
why is phenotypic selection preferred over screening
screening is enormously laborious and time-consuming, a large number of colonies must be screened
27
how does phenotypic selection work
growth conditions where only desired mutants can grow are provided
28
when is phenotypic selection used
used for selectable mutations, there is a growth advantage
29
what are the steps of replica plating
- prepare a spread plate of a population of cells and form colonies
- make a print of those colonies on velvet fabric placed on a wooden block and secured by an elastic band
- stamp the velvet onto secondary plates of varying mediums
- compare the growth on the replica plates
30
describe patching for finding mutants
- pick colonies from master plate with sterile toothpick
- sequentially inoculate gridded test plates with each picked colony, then incubate
- compare growth on the test plates
31
what are the 4 types of mutations
- silent
- missense
- nonsense
- frame shift
32
define: silent mutation
don't change amino acid sequence and don't have a phenotypic effect
33
define: missense mutation
involve a codon change, different amino acid gets introduced
34
define: nonsense mutation
stop codon is suddenly introduced
35
define: frame shift mutations
involves an insertion or deletion of a nucleotide, amino acid sequences aren't related to the protein at all
36
what is the most common type of mutation in labs
missense mutations
37
define: reversion [mutation]
a mutation that "corrects" a metabolic abnormality back to the wild-type form
38
how are reversions avoided [mutation]
mutant strains with multiple mutations are formed, double and triple auxotroph mutant strands are used
39
who developed the replica plating method
Esther Lederberg
40
what did Esther Lederberg prove with replica plating
spontaneous mutation, mutations arise even without selective pressures
41
what did Luria and Delbruck show
variable resistance to phage infection arises in bacteria without selective pressure
42
how does the growth of mutations compare to their parental strains
mutations continually confer growth advantages
43
what is the purpose of restriction enzymes
cut DNA at a specific recognition
44
what is a special feature of restriction enzyme recognition sites
they are usually palindromic
45
what is the most common recognition site length
six bases
46
what is the use of staggered cuts produced by restriction enzymes
allows for the ligation of DNA
47
what are restriction enzymes always paired with
corresponding modification system
48
what is the purpose of methyltransferase in the context of modification enzymes
protects the DNA from restriction enzyme activity
49
what is the purpose of modification enzymes
they restrict incoming bacteriophage DNA that may be harmful to the host
50
where are methyltransferase and the restriction enzyme often located
close together, often in a single operon
51
what enzyme is responsible for ligating DNA fragments
DNA ligase - reattaches phosphate backbone
52
define: vectors
mobile genetic elements that can be used to get DNA shuttled into a cell for cloning experiments
53
what are the types of vectors
- plasmid vectors
- phage vectors
- cosmids
54
what is the purpose of recombinant molecules
used to clone a bacterial gene of interest
55
how are plasmid vectors usually cloned
the DNA and plasmid are cut with the same restriction enzyme and then are ligated together
56
what are the traits of plasmid cloning vectors for easier gene cloning
- origin of replication
- selectable marker gene
- multiple cloning site
- small size
- high copy number
57
what is the purpose of the origin of replication
allows the plasmid to replicate within a host
58
what is the purpose of the selectable marker gene
ensure recipient cells have the plasmid of interest
e.g. antibiotic resistance
59
what is a multiple cloning site
a site that contains many enzyme recognition sites side-by-side
60
what does it mean for plasmids to be in the same Inc group
plasmids that are incompatible with one another and are closely related, replication is controlled by the same genes
61
which gene gets disrupted in blue-white screening
lacZ' gene - codes for one-half of β-galactosidase
62
what is used to test for β-galactosidase activity
X-gal
63
where is the lacZ omega subunit encoded (blue-white screening)
E. coli genome
64
why are alternate hosts sometimes needed for cloning
toxicity issues or codon biases
65
what are shuttle-vector plasmids
vectors with multiple types of origins or replication, expanding the range of hosts they can be inserted into
66
what else is needed in addition to multiple origins or replication in shuttle-vector plasmids
multiple selectable markers
67
what is plasmid replication rate governed by
the origin of replication (oriV) sequence
68
what colour are colonies with an insert successfully added in blue-white screening
white, β-galactosidase is disrupted
69
how do phage vectors work
mix viral DNA with a fragment of interest
70
around how large of a fragment can lysogenic lambda phages carry
~20-kb fragments
71
why do lysogenic lambda phages become lytic after DNA of interest is added
the part that codes for the lysogenic pathway gets replaced
72
what is a drawback to using phage vectors
the host cells lyse and die in the process
73
define: cosmids
phage genomes that omit nearly all the phage DNA, leaving more room for the fragment
74
what portion of the phage genome is left in cosmids
the cos site
75
how big does the cosmid vector + insert have to be
~50 kb
76
what elements are present in cosmids
- cos site
- oriV
- multiple cloning site
- antibiotic selection marker
77
how large can the fragments be in cosmids
35-45 kb
78
what are 3 methods of DNA transfer
- transformation
- conjugation
- transposition
79
describe: transformation [DNA transfer]
introduction of naked extracellular DNA directly into an organism
80
does transformation require cell-to-cell contact [DNA transfer]
it does not
81
define: natural competence
the ability for bacteria to take up DNA directly from the environment
82
what is free DNA used for in bacteria
- source of nutrients
- repairing DNA
- recombination to provide new traits
83
what is a risk of recombination with free DNA
deleterious traits can also be acquired
84
what happens to a DNA strand brought into a naturally competent cell
- it's introduced single-stranded
- other strand of DNA is digested
- single-stranded DNA is coated with the RecA protein
85
why is free DNA introduced as a single strand
it's protected from restriction enzymes
86
why is the single-stranded DNA coated with the RecA protein [transformation, DNA transfer]
it initiates the SOS pathway regulon, DNA introduces itself into the genome through homologous recombination
87
what is required in the host genome for transformation [DNA transfer]
a region of homology
88
what are two ways to artificially induce competence in bacteria
- treatment with calcium cations
- electroporation
89
define: conjugation
the transfer of DNA from cell to cell via direct contact/sex pilus formation
90
what is the F plasmid
- "fertility plasmid"
- best-studied plasmid that encodes for conjugation
91
what genes are critical for mediating the conjugation process
tra region
92
what does the tra gene encode for
production of a mating bridge for transferring plasmid DNA in a specific direction
93
what gets transferred first in conjugation
oriT, the origin of transfer
94
what are F+ cells
E. coli with the F plasmid
95
what cells do F+ cells conjugate with
F- cells
96
what ensures that F+ cells don't conjugate with other F+ cells
tra genes
97
what happens to an F- cell one it has been conjugated with
it becomes a F+ cell
98
how does the F+ cell retain the F plasmid
it replicates itself while mating, and threads one strand into the recipient cell
99
how does F plasmid occasionally integrate into the genome of E. coli
by homologous recombination
100
define: episome
DNA that can integrate into the chromosome but also exist autonomously
101
what are some examples of episomes
- F plasmid
- lysogenic phages
- insertion sequences
- transposons
102
how does the transfer of DNA work with F plasmid genomes
- begins with a portion of the plasmid because of the origin of transfer
- genome itself starts passing through the mating bridge after
- very last bit of DNA passing through would be the rest of the plasmid
103
what is the name of a bacteria cell in which the F plasmid is integrated into the genome
Hfr strain - "High-Frequency of Recombination"
104
how can Hfr stains be used to map genomes
Hfr stains were allowed to mate with recipients for extended periods of time before being disrupted by a blender
105
who discovered using Hfr stains for genome mapping
Joshua Lederberg
106
how can E.coli go from being Hfr to F+
the integrated F factor can excise itself from the genome by homologous recombination
107
how is it possible for F' to contain chromosomal DNA for E. coli
by homologous recombination, when the integrated F factor gets excised, some genomic DNA is excised as well
108
how does triparental conjugation work
- helper plasmid contains tra genes
- helper plasmid conjugates into donor strain
- helper plasmid encodes proteins needed for transfer of recombinant plasmid from donor strain
109
what does transposition involve
movement of insertion sequences and transposons
110
what are some moves DNA can make through transposition
- one plasmid to another
- within and between genomes
- between genomic DNA and plasmids
- from a plasmid back to the genome
111
define: insertion sequence
mobile genetic element that only encodes the proteins needed for transposition
112
define: transposons
mobile genetic elements that contains genes in addition to those needed for transposition
113
does transposition depend on homologous recombination
no, sequence homology is not required
114
what is required in insertion sequences
transposase gene with inverted repeat (IR) regions
115
how long are IR regions
~20 bases
116
what does the resolvase gene do
it recognizes the res site in the transposon
117
describe: replicative transposition
copies the genetic element and moves the copy to another location
118
describe: non-replicative transposition
cuts the genetic element and moves the excised element to another location
119
which form of transposition is resolvase responsible for
replicative transposition
120
what can be used to generate mutants (aside from UV light)
transposons
121
what is the purpose of suicide vectors
delivering transposons to recipient cells
122
define: transposon mutagenesis
the creation of mutants by randomly embedding a transposon in the genome
123
define: transducing particles
viral particles that contain host DNA instead of, or in addition to, viral DNA
124
describe: generalized transduction
- virus accidentally packages a fragment of host cell DNA
- virus delivers random host genome fragment instead of viral DNA to the next cell
- virus is unable to replicate
- few transducing phages
- homologous recombination must occur to integrate into a recipient genome
125
describe: specialized transduction
- lysogenic bacteriophage integrates at specific site
- virus excises incorrectly, includes some host genome
- all virus particles are transducing phages following lysis
- homologous recombination must occur to integrate into a recipient genome
126
what was used to map bacterial genomes prior to DNA sequencing
co-transduction frequency
127
is there phage DNA in transducing particles for generalized transduction
no, it only contains the host DNA
