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Microb 230 Unit 3 > Unit 3.2: Genetic Engineering > Flashcards

Unit 3.2: Genetic Engineering Flashcards

1
Q

Briefly describe biotechnology.

A

the use of microorganisms, cells, or cell components to make a product

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2
Q

List some uses of biotechnologies.

A

Foods, antibiotics, vitamins, enzymes

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3
Q

The name or phrase for:
the insertion or modification of genes to produce desired proteins

A

Recombinant DNA (rDNA) technology

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4
Q

Regarding rDNA, define vector.

A

self-replicating DNA molecule used to transport foreign DNA into a cell

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5
Q

Regarding rDNA, define clone.

A

population of genetically identical cells arising from one cell; each carries the vector

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6
Q

Describe the steps of a typical genetic modification procedure.

A
  1. vector isolated
  2. DNA cleaved into fragments
  3. desired gene selected, inserted into plasmid
  4. plasmid uptake by cell
  5. cells cloned
  6. harvest (either gene or product of gene)
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7
Q

Regarding biotechnology, describe selection.

A

selecting for a naturally occurring microbe that produces a desired product

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8
Q

Regarding biotechnology, what is the usefulness of mutagens?

A

Mutagens cause mutations that might result in a microbe with a desirable trait

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9
Q

Regarding biotechnology, what is the name or phrase for: a targeted and specific change in a gene?

A

Site-directed mutagenesis

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10
Q

Describe the natural function of restriction enzymes.

A

special class of DNA-cutting enzymes that protect a bacterial cell by hydrolyzing phage DNA

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11
Q

Describe how bacterial DNA is not digested within a bacterial cell.

A

The bacterial DNA is protected from digestion because the cell methylates (adds methyl groups to) some of the cytosines in its DNA.

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12
Q

What is the important feature of a restriction enzyme for rDNA techniques?

A

only one particular sequence of nucleotide bases in DNA is cut, and it cuts this sequence the same way each time

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13
Q

Name and describe the two cuts a restriction enzyme can make.

A
  • blunt end - straight across
  • sticky end - staggered cuts
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14
Q

Why is the sticky cut of a restriction enzyme named this way?

A

The sticky ends “stick” to stretches of single-stranded DNA (by complementary base pairing).

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15
Q

Describe the role of a restriction enzyme making a sticky cut in making rDNA.

A

When two fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing.

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16
Q

After two sticky cuts come together and form a new molecule, what happens next?

A

DNA ligase is used to unite the two backbones of the two fragments resulting in a single DNA molecule.

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17
Q

What two things are typically used as vectors in biotechnology?

A

plasmids and viruses

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18
Q

Regarding biotechnology, describe shuttle vectors and their usefulness.

A
  • they can move between several difference species
  • good for moving cloned DNA sequences between organisms
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19
Q

Describe polymerase chain reaction.

A

a technique by which small samples of DNA can be quickly increased to quantities that are large enough to be useful

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20
Q

Briefly identify the diagnostic tests for which a polymerase chain reaction is used.

A
  • genetic diseases and
  • detecting pathogens
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21
Q

Briefly contrast reverse-transcription PCR.

A

uses mRNA as template instead of DNA

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22
Q

In preparation for PCR, what components are supplied?

A
  • target DNA
  • primers
  • the four nucleotides
  • DNA polymerase
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23
Q

Regarding PCR, describe the primers.

A

Short pieces of nucleic acid complementary to the ends of the target DNA that help start the reaction.

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24
Q

Regarding PCR, after the primers attach, what reaction occurs next?

A

the polymerase synthesizes new complementary strands

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25
Q

Regarding PCR, after the new copy is made, what is the purpose of the next application of heat?

A

the DNA is heated to convert all the new DNA into single strands

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26
Q

What device is used to perform the PCR and what is its function?

A

The thermal cycler can be set for the desired temperatures, times, and number of cycles.

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27
Q

Name the five ways we can get rDNA into cells.

A
  • transformation
  • electroporation
  • protoplast fusion
  • gene gun
  • microinjection
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28
Q

Regarding biotechnology, briefly describe transformation.

A

a procedure during which cells can take up “naked” DNA from the surrounding environment (a solution)

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29
Q

Briefly describe electroporation.

A

a procedure where electrical current forms pores in
cell membranes

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30
Q

Briefly describe Protoplast fusion.

A

Removing cell walls from two bacteria allows so they can fuse.

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31
Q

Regarding protoplast fusion, how are protoplasts formed?

A

bacterial cell walls are enzymatically digested, producing protoplasts

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32
Q

Regarding protoplast fusion, protoplasts are treated with what before they fuse?

A

polyethylene glycol

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33
Q

Regarding protoplast fusion, after the protoplasts fuse, what happens next?

A

segments of the two chromosomes recombine

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34
Q

Regarding protoplast fusion, after the chromosomes recombine, what happens next?

A

the new recombinant cell regrows its cell wall

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35
Q

Briefly describe the “bullets” and “propellant” of the gene gun.

A
  • Microscopic particles of tungsten or gold are coated with DNA and
  • propelled by a burst of helium through the plant cell walls.
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36
Q

Describe the physical requirements of the microinjection technique to get rDNA into animal cells.

A

This technique requires the use of a glass micropipette with a diameter that is much smaller than the cell.

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37
Q

Briefly describe the requirements of getting foreign DNA to survive in a cell.

A

Foreign DNA will survive only if it’s either
* present on a self-replicating vector or
* incorporated into one of the cell’s chromosomes by recombination.

38
Q

Briefly describe a genomic library.

A

Collections of clones containing different DNA
fragments.

39
Q

Briefly describe the process of creating a genomic library from a particular organism.

A
  • the DNA is digested by restriction enzymes,
  • the restriction fragments are then spliced into plasmid or phage vectors,
  • the recombinant vectors are introduced into bacterial cells.
40
Q

What is the most basic requirement for the successful creation of a genomic library?

A

At least one clone exists for every gene in the
organism

41
Q

What enzyme is used to resolve the issue of introns in cloning genes in eukaryotic organisms?

A

reverse transcriptase

42
Q

What is the product of reverse transcriptase?

A

an artificial gene of complementary DNA that contains only exons

43
Q

What is reverse transcriptase?

A

an enzyme that synthesizes complementary DNA from an mRNA template

44
Q

After reverse transcription creates a strand of complementary DNA, what happens next?

A

the mRNA is digested away allowing for DNA polymerase to come in and synthesize its complement (to form a double-stranded piece of DNA).

45
Q

What is the significance of reverse transcriptase targeting mRNA?

A

mRNA has the introns removed, coding only for the
protein product

46
Q

Briefly describe synthetic DNA.

A

genes built using a DNA synthesis machine

47
Q

What is the purpose of blue-white screening?

A

To identify colonies that have picked up the recombinant plasmid.

48
Q

When performing blue-white screening, which color indicates colonies with the desired gene.

A

Bacteria that picked up the recombinant plasmid will not hydrolyze lactose and will produce white colonies.

49
Q

What is the gene in blue-white screening that is responsible for the blue or white color? And what does the gene produce?

A
  • the lacZ gene
  • enzyme β-galactosidase
50
Q

What is the antibiotic used for blue-white screening?

A

ampicillin

51
Q

What is the substrate for β-galactosidase used in blue-white screening?

A

X-gal

52
Q

Regarding selecting a clone, describe colony hybridization.

A

Use of DNA complimentary to the target gene to detect its presence.

53
Q

Regarding colony hybridization, describe a DNA probe.

A

single-strand DNA “labeled” with a detectable enzyme or dye

54
Q

After a master plate has been made, what are the steps of colony hybridization?

A
  1. nitrocellulose filter
  2. lyse bacteria with detergent
  3. separate strands with sodium hydroxide (NaOH)
  4. add probes
  5. hybridization occurs
  6. wash to remove unbound probes
  7. compare to master plate
55
Q

Regarding biotechnology, what are the advantages and disadvantages of using E. coli?

A

– Advantages: easily grown and its genomics are
known
– Disadvantages: produces endotoxins and does not
secrete its protein products

56
Q

Regarding biotechnology, what are the advantages of Saccharomyces cerevisiae?

A

– Easily grown and has a larger genome than bacteria
– Expresses eukaryotic genes easily

57
Q

Regarding biotechnology, what are the advantages of Plant cells and whole plants?

A

– Express eukaryotic genes easily
– Plants are easily grown, large-scale, and low-cost

58
Q

Regarding biotechnology, what are the advantages and disadvantages of Mammalian cells?

A

– Express eukaryotic genes easily
– Can make products for medical use
– Harder to grow

59
Q

Name several therapeutic applications of DNA technology.

A

pharmaceuticals
subunit vaccines
DNA vaccines
Gene therapy
Gene editing
Gene silencing

60
Q

Briefly describe subunit vaccines.

A

vaccine created in yeast modified with only a portion of the proteins from a pathogen

61
Q

Briefly describe DNA vaccines.

A

Nonpathogenic viruses carrying genes for pathogen’s
antigens.

62
Q

Briefly describe gene therapy.

A

Replacement of defective or missing genes.

63
Q

Briefly describe gene editing.

A

using CRISPR to correct genetic mutations at specific locations

64
Q

Describe gene silencing.

A
  • small interfering RNAs are formed which
  • bind to mRNA which are then
  • destroyed by RNA-induced silencing complex proteins.
65
Q

Name and describe the new technology to perform gene silencing.

A

RNA interference
* inserting DNA that encodes small interfering RNAs

66
Q

Describe shotgun sequencing (of a genome).

A

A genome is cut into pieces, and each piece is sequenced. Then the pieces are fit together using a computer.

67
Q

Describe Metagenomics

A

the study of genetic material directly from environmental samples

68
Q

What is the The Human Proteome Project?

A

maps proteins expressed in human cells

69
Q

Name several scientific applications of DNA technology.

A
  • bioinformatics
  • proteomics
  • reverse genetics
  • Southern blotting
  • genetic testing
70
Q

Briefly define bioinformatics.

A

understanding gene function via computer assisted analysis

71
Q

Briefly define proteomics.

A

determining proteins expressed in a cell.

72
Q

Briefly describe reverse genetics.

A

discovering gene function from a gene sequence

73
Q

Briefly describe Southern blotting.

A

DNA probes detect specific DNA
in fragments separated by gel
electrophoresis
* restriction fragment length polymorphisms

74
Q

Briefly describe genetic testing.

A

screening of parental or fetal tissue for several hundred possible genetic diseases

75
Q

What is the first step of Southern blotting?

A

DNA of interest is cut into fragments (RFLPs).

76
Q

Regarding Southern blotting, the name or phrase for the fragments.

A

Fragments are called restriction fragment length polymorphisms, or RFLPs (pronounced “rif-lips”).

77
Q

Regarding Souther blotting, after making the fragments, what comes next?

A

The fragments are separated into bands according to size by gel electrophoresis. (The bands can be made visible with staining.)

78
Q

Regarding Southern blotting, what comes after forming the bands?

A

The bands are blotted onto a nitrocellulose filter.

79
Q

Regarding Southern blotting, what comes after the bands are transferred to the filter?

A

The filter is exposed to DNA probes.

80
Q

Regarding Southern blotting, what is revealed by the DNA probes?

A

The fragment containing the gene of interest is identified by a band made visible on the filter.

81
Q

Regarding forensic microbiology, what is the use of DNA fingerprinting?

A

to identify bacterial and viral pathogens

82
Q

Describe DNA fingerprinting.

A

Using DNA chips or PCR microarrays to screen a sample for multiple pathogens at once.

83
Q

Name two key requirements of forensic microbiology used in a court of law that are more strict than for the medical community.

A

– Proper collection of evidence
– Establishing a chain of custody

84
Q

Define nanotechnology.

A

Making molecular or atom-sized products.

85
Q

Regarding nanotechnology, describe the usefulness of bacteria.

A
  • can provide the needed small metals without producing the toxic waste
  • some bacteria can make nanoparticles
86
Q

What is the natural source of Ti plasmids?

A

occurs in bacteria Agrobacterium tumefaciens

87
Q

What is the natural effect of Ti plasmids on infected plants?

A

the Ti plasmid causes the formation of a tumorlike growth called a crown gall

88
Q

What is the biotechnology application of Ti plasmids?

A

Can be used to introduce rDNA into a plant

89
Q

Identify several agricultural applications of biotechnology.

A
  • Bt toxin
  • Herbicide resistance
  • Suppression of genes
    – Antisense DNA
  • Nutrition
  • Human proteins
90
Q

Identify some safety issues and ethical concerns of using DNA technology.

A
  • Need to avoid accidental release into the environment
  • Genetically modified crops must be safe for
    consumption and for the environment
  • Who will have access to an individual’s genetic
    information?

Microb 230 Unit 3 (3 decks)

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