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Microbiology > test 3 > Flashcards

test 3 Flashcards

1
Q

epigenetic Control

A

methylating nucleotides to turn off

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2
Q

post-transcriptional control

A

microRNA: degrade RNA; double stranded RNA prevents production of a protein

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3
Q

other mechanisms for regulatory control

A

epigenetic control, post-transcriptional control

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4
Q

how do mutations occur?

A

substitutions, insertions or deletions

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5
Q

types of point mutations

A

missense, nonsenese, frameshift

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6
Q

which point mutation is most lethal?

A

frameshift, because it is related to an insertion or deletion of a base

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7
Q

missense mutation results in

A

new AA

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8
Q

nonsense mutation results in

A

stop codon

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9
Q

rate of mutation rates in the absence of mutagens

A

base pairs: 1 in 10^9

replicated genes: 1 in 10^6

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10
Q

types of mutagens

A

chemicals, radiation, nucleotide excision repair

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11
Q

nitrous acid

A

chemical, byproduct of fossil fuel burning, oxidized adenine and binds with cytosine

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12
Q

intercalating agents:

A

extra bases added in between bases

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13
Q

types of radiation

A
  1. ionizing: (X-rays; gamma rays)

2. non ionizing

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14
Q

ionizing radiation

A

xrays and gamma rays: cause formation of ions which oxidize nucleotides and breaks deoxyribose phosphate

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15
Q

postive (direct) selection

A

detects mutant cells because they grow or appear different

-plate bacteria on a medium that allows mutant to grow but not (penicillin)

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16
Q

negative (indirect) selection

A

detects mutant cells because they do not grow or cannot perform a function

replica plating
grow in plate with his and plate without his nd you look for absence of organism

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17
Q

auxtotroph

A

mutant with nutritional requirement absent in parent

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18
Q

vertical gene transfer

A

transfer of genes from parent to offspring

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19
Q

types of horizontal gene transfer (between same generation)

A

transformation, conjugation, transduction

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20
Q

tranformation

A

naked DNA is picked up from the environment

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21
Q

what experiment showed transformation

A

Griffith’s experiment with the mouse and encapsulated bacteria

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22
Q

types of plasmids

A
  1. conjugative plasmid
  2. dissimilation plasmid
  3. R factors
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23
Q

conjugative plasmid

A

carriers genes fro sex pili abd transfer of plasmid

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24
Q

dissimilation plasmids

A

encode enzymes for catabolism of unusual compounds

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25
Q

r factors

A

encode for antibiotic resistance

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26
Q

another type of DNA that makes pathogens resistant

A

transposons

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27
Q

transposons

A

segments of DNA that can move from one region of DNA to another

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28
Q

insertion sequence:

A

contains recognition sequences and transposase for cutting and resealing DNA

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29
Q

complex transposons

A

carry other genes (antibiotic resistant genes)

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30
Q

biotechnology

A

use microorganism, cells, cell components to make products like enzymes, food, antibiotics

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31
Q

recombinant DNA technology

A

artificial techniques (lab based) for making recombinant DNA protein —> DNA manipulation

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32
Q

how do we get a Bacterium with a specific trait?

A

Select for a naturally occurring microbe that has trait or produces desired trait; use mutagens to introduce MUTATION that might result in a microbe with desired trait; or use SITE DIRECTED MUTAGENESIS: (single base change in DNA)- CRISPR

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33
Q

typical genetic modification procedure

A
  1. need vector (plasmid) 2. Have DNA of interest (cut gene with enzyme) 3. introduce gene to vector 4. plasmid is taken up by the cell 5. two goals in mind: -create and harvest copies of gene -create and harvest protein product of gene
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34
Q

how do we obtain desired/foreign DNA?

A

genomic libraries

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35
Q

genomic libraries

A

collections of clones containing different DNA fragments, they contain every gene in organism, stored in plasmids or phages(insert larger pieces )

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36
Q

what is the problem when we are generating eukaryotic libraries?? and what is the solution

A

Dna contains introns and exons, so we use mRNA as template to produce Complementary DNA (cDNA) using transcriptase

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37
Q

what enzyme is used to make RNA go to cDNA

A

transcriptase

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38
Q

cDNA library

A

contains cDNA produced from mixture of all mRNAs from tissue or cell type

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39
Q

what is the purpose of PCR

A

to make more copies of DNA

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40
Q

what is PCR used for?

A

clone DNA for recombination, Amplify DNA to decidable levels, Sequence DNA, diagnose genetic diseases, detect pathogens

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41
Q

steps to PCR

A
  1. denaturation: single stranded DNA, heat is used
  2. Annealing: primer specific to gene we want
  3. Extension: Dna pol comes in at 3 prime end and adds nucleotides

process repeated to make make many copies of target gene, proceeds exponentially

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42
Q

why is a primer important in PCR?

A

picks a certain gene

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43
Q

what needs kind of DNA pol has to be used in PCR

A

one resistant to high temperatures

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44
Q

what tool is used for manipulating DNA?

A

a vector, carries gene to another cell

self replicating molecule

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45
Q

what a shuttle vector?

A

can exist in several different species

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46
Q

types of vectors

A

plasmids, lambda phages

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47
Q

what are the characteristics for a good vector?

A

must have an origin of replication, selectable manner, multiple cloning sites

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48
Q

restriction enzymes

A

very specific and recognize 4- 6- and 8- palindromic sequences

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49
Q

restriction enzymes EcoRI

A

cit between G and A

50
Q

what is the role os restriction enzymes in making recombinant DNA?

A

as long as they are cut by the same enzyme, they can be put together, then DNA ligase will put the cDNA together

51
Q

what happens after recombination?

A

Transfection

52
Q

transfection

A

process of introducing naked DNA into living cell. may be physical or chemical

53
Q

types of transfection

A

transformation, electroporation, Photoplast fusion, microinjection, gene gun

54
Q

transformation transfection

A

e.coli soaked in CaCl2 and make them competent (able to take up external DNA
put them in ICE and put them at 45 Celsius, then suck up DNA into cell (heat shock)

55
Q

electroporation Transfection

A

uses machine that use electrical current that make holes in membrane and DNA goes in

56
Q

photoplast fusion

A

not commonly used in bacteria, but when do, have to treat with lysozyme and cell wall is gone

57
Q

gene gun

A

used in plants

58
Q

microinjection

A

:)

59
Q

purpose of blue white selection

A

bacteria spread on plate containing Xgal, with the help of Lac Z you are able to which bacteria breaks down Xgal. the one that doesn’t break down the protein will be the recombinant DNA

60
Q

prokaryotes used in genetic engineering? which one is best and why?

A

E.coli and Bacillus Subtilis

B. subtillis is because it secretes products a spits it out of the cell.

E. coli is not preferred because cells lysed to get product and products may contain endotoxin

61
Q

types of eukaryotic cells used for genetic engineering?

A

Saccharomyces cerevisiae(yeast), plant cells, mammalian and human cells

62
Q

saccharomyces cerevisiae (yeast)

A

they are easy to grow, genome is known, products secreted

63
Q

plant cells

A

easily grown, low cost, large scale

64
Q

mammalian/human cells

A

harder to grow, make products for medical use (hormones, cytokines, interferons)

65
Q

genetically engineered human products

A

insulin; subunit vaccines (viral coat-hepatitis B); DNA vaccines (cloning genes for use as vaccines (HIV)

66
Q

Gene therapy

A

replace defective or missing genes (hemophilia, sickle-cell)

67
Q

gene silencing use

A

RNAi interference for cancer treatment and viral infections

68
Q

how does gene silencing work

A

RNAi bind to mRNA and prevents translation

mRNA destroyed by RNA-induced silencing complex (RISC)

69
Q

RNA-induced silencing complex (RISC)

A

destroys mRNA

70
Q

scientific applications:

A

shotgun sequencing, metagenomics, human genome project, human proteome project, bioinformatics, reverse genetics, proteomics

71
Q

shot gun sequencing

A

sequences small pieces of genomes then assembled by a computer

72
Q

metagenomics

A

study of genetic material directly from environmental samples

73
Q

human genome project

A

sequenced entire human genome

74
Q

human proteome project

A

map proteins expressed in human cells

75
Q

bioinformatics

A

using computer to analyze gene function

76
Q

reverse genetics

A

protein/ mRNA —> gene (gene function from sequence)

77
Q

proteomics

A

determining proteins expressed in a cell (different genes expressed)

78
Q

how are Ti plasmids used and why are they useful?

agriculture

A

they use tumor inducing plasmid which integrate Into the plant genome and causes tomor-like growth.

may help with pest resistant plants, herbicide resistant plants, suppression of genes, animal growth hormones

79
Q

what Bacteria produce Ti plasmids?

A

in Agrobacterium Tumefaciens

80
Q

example of suppression of genes

A

MacGregor tomatoes: antisense DNA blocks lectin degradation, you can shelf for longer

81
Q

examples of animal growth hormone produced by E.coli

A

Bovine: increase weight gain or milk production

swine: increase weight in swine

82
Q

what an issue regarding bioTech

A

can’t show that something is safe under all conceivable conditions

83
Q

safety standards of biotech

A

avoid accidental release

genetically modified crops must be safe for consumption and for environment

84
Q

moral and ethical issues of biotech

A

who will have access to an individuals genetic info

human cloning, stem cells and baby engineering

85
Q

taxonomy

A

science of classifying organisms

-shows degree of similarity organisms

86
Q

systematics or phylogeny

A

study of the evolutionary history of organisms

87
Q

organisms are….

A

product of their genetic story
give rise to like organisms
each species retains some characteristics of its ancestor
led to suggestion that all organisms evolved from a common ancestor

88
Q

taxonomy is useful for

A

classifying organisms into groups, reference for identifying organisms, and provides a common ahem for organisms (nomenclature)

89
Q

common properties to group organisms

A
  1. composed of cells surrounded by plasma membrane
  2. use ATP for energy
  3. DNA
90
Q

what was the old method for grouping organisms?

A

classification based on morphology and physiology

fossils

91
Q

New method for grouping organisms?

A

comparison of molecular sequences

92
Q

how do we identify microorganisms?

A

matching characteristics of an “unknown” organism to lists of known organisms

93
Q

how do we classify microorganisms?

A

by placing organisms into groups of related species

lists of characteristics of known organisms

94
Q

identification methods:

A

morphological characteristics, differential staining, biochemical tests, serological testing, Phage typing, fatty acid profiles, flow cytometry, DNA base composition, DNA fingerprinting, Nucleic acid amplification tests, ribotyping-RNA sequencing , nucleic acid hybridization , fluorescent In Situ Hybridization

95
Q

morphological characteristics

A

shape, size, endospore production, flagella

96
Q

differential staining

A

gram staining, acid fast staining

-based on chemical composition of the cell wall

97
Q

biochemical tests

A

determines the presence of specific enzymes

98
Q

rapid identification methods

A

perform several biochemical tests simultaneously

ex: enterotube can test 15 biochemical tests

99
Q

serological testing

A

involves combining known antiserum(antibody) against unknown bacterium

can differentiate between species and strains within species

antibodies react very specific to antigens

100
Q

types of serological testing

A

slide agglutination

ELISA

western Blot

101
Q

slim agglutination

A

use antibody against a specific cell

+ test= will react - test = does not react

102
Q

ELISA

A

TELLS how much of the organism is present

bottom of the of the tube are the antibodies to organism that we are trying to detect, then wash, then add more antibodies with enzyme, if substrate is broken down then color change

color change indicates + for organism in mind

103
Q

western blot

A

to test for Lyme disease…

isolate protein of interest (one that causes Lyme disease), then proteins are loaded on gel where they are separated by charge by electrophoresis, then washed with patients serum, followed by an antihuman antibody with enzyme, then if the test is positive, the spot will be visible and the patient will be + for the disease

104
Q

phage typing

A

uses virus to identify bacteria because only certain viruses are able to infect certain bacteria

clearing on plate will indicate which bacteria was lysed by the virus

useful for tracing origin/cause of disease

105
Q

Fatty acid profiles

A

Fame: fatty acid methyl Esters provide profiles that are constant for a particular species

106
Q

flow cytometry (expensive machine)

A

uses differences in electrical conductivity between species or fluorescence

provide info about shape, size, density, and surface without culturing

107
Q

DNA base composition

A

when 2 organisms have the same GC content = organisms may be related

108
Q

how of you calculate DNA base composition?

A

total CG/ by total bases = 6/10 = 60%

109
Q

DNA finger printing

A

electrophoresis of restriction enzyme digested DNA

more similar pattern, the more closely related the organism
separated by size

110
Q

Nucleic Acid amplification tests

A

use PCR to amplify DNA of unknown microorganisms to detectable levels

uncultivable organisms

111
Q

ribotyping - rRNA sequencing

A

advantage: all cells have ribosomes, rRNA is highly conserved, no culturing required (PCR using universal primers)

112
Q

nucleic acid hybridization (blot and DNA chip)

A

measures ability of DNA strands From one organism to hybridize with DNA strands of another organism

southern blot= DNA to DNA

Northern Blot = DNA to RNA / RNA to RNA

113
Q

southern blot

A

heat DNA from each organism, then mix DNAs , if complete hybridization, them completely related

114
Q

nucleic acid hybridization useful in colony blot

A

will use probe to detect gene out of a mix of genes

gene of interest is labeled, gene of interest bacteria will survive after substrate added

– tells you which bacterial clone has gene of interest

115
Q

DNA chip

A

take sample, get DNA, add fluorescent tag, sample on fluorescent chip, if complementary then colorful

116
Q

Fluorescent In Situ Hybridization (FISH)

A
  • fluorescent RNA/DNA probes stain targeted microorganisms
  • probe is single stranded
  • determines identity, abundance, and activity of microorganisms in an environment
117
Q

what are the 2 ways classification methods are put together?

A

dichotomous keys and cladograms

118
Q

dichotomous keys

A

flow chart that leads to identification,

no phylogeny info

119
Q

cladogram

A

phylogenetic tree,

  • shows evelotionary
  • uses highly conserved genes
  • branching patterns show which species share more recent common ancestry
  • branch length represent the amount of change
120
Q

Bergey’s Manual of Determinative bacteriology

A

morphology, differential staining , biochemical test s

121
Q

Bergey’s manual systematic bacteriology

A

based on rRNA sequencing

Microbiology (8 decks)

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