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Microbiology > Lecture 11 - Biotechnology > Flashcards

Lecture 11 - Biotechnology Flashcards

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Biology 101 flashcards
1
Q

what is biotechnology?

A

the use of microbiological and biochemical techniques to produce useful products and solve practical problems

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2
Q

how is biotechnology used in medicine, industries, and forensics?

A

Medicine: diagnosis of diseases.
Industry: production of insulin.
Forensics: identifying criminals.

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3
Q

what two things of biotechnology are we focusing on?

A

Identification technologies and DNA cloning

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4
Q

what are the different types of identification technologies?

A

-DNA sequencing
-DNA probes (including colony blotting, FISH, DNA microarrays)
-DNA fingerprinting (including RFLP and PCR)

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5
Q

what is DNA sequencing within identification technologies?

A

determining the exact order of base pairs in a segment of DNA

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6
Q

what is an example of DNA sequencing

A

The human genome project, when there’s 3 billion base pairs but only around 25,000 genes

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7
Q

why do we need all those extra base pairs? aka why do we need the non-coding DNA?

A

we need the non-coding DNA because it is important since we need these extra base pairs to code for tRNA, rRNA, promoter, operator, introns, etc. All these are needed for gene regulation.

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8
Q

what is DNA sequencing used to identify? what is it essential for?

A

DNA sequencing is used to identify microbes in nature, including unculturable ones. DNA sequencing is essential for biotechnological applications, ex. to design DNA probes.

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9
Q

what are DNA probes?

A

short, labeled single-strand bases that are used to detect a complementary sequence of DNA

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10
Q

what are examples of what DNA probes do?

A
  • To look for the breast cancer gene within the whole human genome.
  • To identify the pathogen within the patient’s sample.
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11
Q

what are 3 examples of how we use DNA probing?

A
  1. Colony blotting (cultured organisms)
  2. Fluorescence in situ hybridization - FISH. (non-cultured organisms)
  3. DNA microarray
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12
Q

what are the steps of colony blotting?

A
  1. Colonies are placed on an agar plate.
  2. Colonies are transferred in place (“blotted”) to a nylon membrane.
  3. The membrane is soaked in an alkaline solution to lyse the cells and denature their DNA.
  4. The probe is added that binds to the DNA of interest.
  5. By locating the positions to which the probe has bound, colonies that have that specific DNA of interest can be located.
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13
Q

is colony blotting cultures organisms?

A

yes

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14
Q

what is FISH?

A

Fluorescence in site hybridization, non-cultured organisms.
It helps to determine the presence or absence
of specific DNA sequences.
One probe can be specific for archaea, and another for bacteria.

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15
Q

what is DNA microarray?

A

DNA microarray is a technique that detects many genes at one time. It uses a chip with wells, where each well contains a specific DNA probe

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16
Q

what is DNA fingerprinting?

A

the isolation and visualization of DNA sequences to help identify an unknown (you isolate the DNA from the cell)

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17
Q

what are the two techniques of DNA fingerprinting?

A
  1. Restriction fragment length polymorphisms (RFLP’s)
  2. Polymerase chain reaction (PCR)
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18
Q

what are RFLP’s?

A

they are restriction fragment length polymorphisms that cut the DNA. It is a creation of fragments of different lengths, by using restriction enzymes to cut the DNA out

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19
Q

how do restriction enzymes work?

A

they cut the DNA at specific 4-6 base pair sequences. They are essentially scissors that cut out specific DNA pairs, giving you fragments of DNA

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20
Q

how do we visualize the fragments that restriction enzymes cut up?

A

by using gel electrophoresis

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21
Q

what is the charge of DNA?

A

negative charge

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22
Q

what is the DNA gel stained with? what does it do?

A

The DNA gel is stained with ethidium bromide, a dye that fluoresces under UV light.

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23
Q

where does the DNA go towards?

A

the positive charge

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24
Q

what does the gel help to do in DNA fingerprinting?

A

the gel helps to separate the fragments based on size.

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25
Q

what do we compare the fragments with?

A

we use the standard: molecular weight ladder to compare the fragments to it

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26
Q

what size fragment moves the fastest toward the positive charge?

A

the smaller size, so the smaller kb

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27
Q

what is PCR?

A

polymerase chain reaction

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28
Q

what is the buzzword for PCR?

A

amplification

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29
Q

what does the PCR do?

A

it amplifies a DNA sequence, using specific primers and a thermal cycler to replicate the DNA

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30
Q

what is a thermal cycler?

A

a machine that helps to replicate the DNA

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31
Q

what does the amplification of the DNA sequence in PCR help us visualize?

A

it helps us visualize amplified DNA on a gel

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32
Q

what are the three steps to amplifying DNA?

A
  1. Denaturation (unzips the DNA) - using primers that are specific for certain genes.
  2. Annealing (the primers sticking to their complementary sequence on DNA) - using DNA polymerase for replication.
  3. Synthesis
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33
Q

why is the band of the PCR-positive patient so thick?

A

it is thick because you have so many fragments of the same size stacked together

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34
Q

What do you do with SARS-CoV-2 PCR testing since its genome is an ss RNA?

A

you would first have to do reverse transcriptase by converting the ss RNA to a ds DNA, then you can begin the process of PCR

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35
Q

which identification technology looks for a gene by trying to amplify it and displaying amplified fragments on a gel?

A

PCR DNA fingerprinting

36
Q

what is DNA cloning?

A

it is a procedure in which a gene of interest is extracted from a source and inserted into a cell, where it can be replicated and expressed

37
Q

what does DNA cloning involve?

A

DNA cloning involves:
-Recombinant DNA (rDNA): this is a new combination of DNA that is formed by joining DNA molecules from two (or more) different sources.
This is called…
-Genetic engineering: to alter the genome of an organism

38
Q

what is an example of DNA cloning?

A

Inserting a gene to make insulin from a human cell into a bacterial cell to mass produce insulin for human use.
How did we get insulin before?
From animals, like pigs.

39
Q

In DNA cloning, what do we take the genome out of? Where do we insert it? What does this make?

A

We take the genome out of the source and insert it into a mass producer, creating a recombinant

40
Q

why do we often use bacteria as a mass producer?

A

because it is easy and quick to grow

41
Q

what is the simplest process of DNA cloning?

A

Going from prokaryote (source) to prokaryote (mass producer).

42
Q

what may the product of DNA cloning be?

A

a protein or a gene/DNA

43
Q

what are the 5 steps of DNA cloning?

A
  1. Obtain DNA
  2. Cut DNA at restriction sites.
  3. Insert gene of interest into vector.
  4. Insert the vector into the host cell (mass producer).
  5. Collect the product.
44
Q

explain step 1 of DNA cloning.

A

To obtain the source DNA.
This is when we take out the gene of interest. We do this by lysing the source cell that contains the gene of interest, and we collect the DNA.

45
Q

explain step 2 of DNA cloning

A

We cut DNA at the restriction sites using restriction enzymes. This removes the genes from the chromosome.
The restriction enzymes create multiple fragments of variable lengths with “sticky” ends.
This allows the fragments to hybridize to the other DNA cut with the same enzyme.

46
Q

in step 2 of DNA cloning, why do we hybridize the fragments to the other DNA cut with the same enzyme?

A

we do this because the complimentary ends need to stick to each other

47
Q

what is step 3 of DNA cloning?

A

when we insert the gene of interest into the vector.

48
Q

what is a vector and what examples of vectors are being used?

A

a vector is anything that carries the gene into the cell. examples of vectors are plasmids or phages

49
Q

why do we use phages and plasmids as vectors?

A

Because they have their own origin of replication and allow the gene to jump species (meaning it has the capacity to enter any bacterial cell)

50
Q

why do we need to cut the vector open with the SAME restriction enzyme?

A

So that the vector and source DNA have complementary ends to stick together.

51
Q

explain step 4 of DNA cloning

A

When we insert the vector into the host cell (the mass producer). We also culture the bacteria in this step.
The hosts are E. coli and Bacillus subtilis.

52
Q

what are the methods of step 4 of inserting the vector into the host cell?

A
  1. Artifical transformation (for plasmids): the cells are made competent artificially for DNA uptake. We can make the cells competent by:
    - Heat shock
    - Electroporation
  2. Artificial transduction (for phages): phage-mediated delivery of DNA into host cells
53
Q

explain step 5 of DNA cloning and what two products are made

A

When we collect the product. The products can be:
Protein:
- We lyse the cells and purify the protein (ex: E. coli).
- We collect and purify the secreted protein from the cells (ex: Bacillus subtilis).
Gene:
- We lyse the cells and collect the gene of interest.

54
Q

what is another variation of DNA cloning other than prokaryote to prokaryote? how do we do this?

A

We can clone eukaryotic genes into prokaryotic genes.
We do this by:
- Extracting mRNA from the source cells.
- Creating a ds cDNA from isolated mRNA using reverse transcriptase and DNA polymerase

55
Q

know exact steps from DNA cloning of prokaryote to prokaryote in picture?

A

??

56
Q

how do we clone genes into eukaryotic hosts? what are the DNA insertion methods?

A

-The host cells are grown in culture
The DNA insertion methods include:
- Artificial transformation
- Viral or bacterial-mediated gene delivery
- Gene gun (in plant cells)
- Microinjection (in animal cells)

57
Q

why is a plasmid needed as a carrier of a gene of interest in gene cloning?

A

because it enables the gene to jump species

58
Q

what are transgenic organisms? what are they also called?

A

organisms that have foreign DNA present in every cell. they are also called genetically modifies organisms (GMOs).

59
Q

what are three ways to add/introduce foreign DNA to plants and what are the benefits?

A
  1. Pest-resistance:
    -Gives Bt toxin gene against insects.
    -Gives viral, bacterial, and fungal resistance.
  2. Herbicide-resistance:
    -Glyphosate (roundup).
  3. Improved nutritional value:
    -Golden rice with B-carotene (produces vitamin A).
    -Canola oil with enhanced vitamin E or fatty acids.
60
Q

how can plants be future GMOs?

A

-Increased shelf life of fruits and vegetables.
-Drought, salt, frost, chemical tolerance.
-Production of biofuels, pharmaceuticals, enzymes, plastics, and rubber.
-Pollution of carbon-capturing plants.
-Nitrogen-fixing crops
-Allergen-free plants
-Low-maintenance lawns
-Caffeine-free coffee beans
-Edible vaccines

61
Q

what are ways we can apply DNA cloning to animals?

A

We can research human diseases using animal models, including:
- OncoMouse
- Fluorescent animals (GFP gene)
- Brainbow mice

We can use it for food production:
-AquAdvantage fish (making the fish bigger)

We can use it for “pharming”:
-Using milk or egg-producing animals to make pharmaceuticals (ATryn - antithrombin goat)

Organ transplants

Disease control:
-Malaria-proof mosquito
-Lysozyme in goat milk for diarrhea control
-Human antibodies from mice

Environmental protection:
-Enviropig

Production of spider silk:
-Spidergoat

62
Q

what are concerns about transgenic organisms?

A

-That they can introduce allergens/toxins/carcinogens.
-That they can change ecology: by killing good insects, creating plant monocultures, and escaping and outcompeting wild counterparts.

63
Q

what is gene therapy in people?

A

it is when you replace a dysfunctional gene with a normal one

64
Q

what cells are altered in gene therapy in people?

A

one dysfunctional cells are genetically altered

65
Q

what are two examples of gene therapy?

A

-Severe combined immunodeficiency (SCID)
-Leber’s congenital amaurosis (LCA: a retinal disease)

66
Q

what part of the cell does gene therapy alter? aka what are we trying to do?

A

gene therapy alters the functioning of cell genetics including:
-melanoma and blood cancers
-HIV

67
Q

is gene therapy licensed?

A

no, it is still very experimental

68
Q

what are new gene editing techniques? what do they do for gene therapy?

A

New gene editing techniques are CRISPR/Cas protein may make the gene therapy process easier

69
Q

what are hopes for gene therapy in the future?

A

future hopes for diseases such as Duchenne muscular dystrophy, cystic fibrosis, sickle cell anemia, hemophilia, and other cancers

70
Q

can we do germ line gene therapy?

A

no

71
Q

which of the following represents use of gene therapy?
- OncoMouse
- Golden rice
- “Pharming”
- Cancer immunotherapy

A

Cancer immunotherapy

72
Q

DNA fingerprinting that amplifies a single gene of interest which is then visualized on a gel is called:

A

PCR

73
Q

what does a gel electrophoresis machine do?

A

Separates DNA sequences by size

74
Q

why would an E. coli make insulin?

A

It was artificially transformed with the insulin gene

75
Q

What is “pharming?”

A

Engineering animals to make pharmaceuticals

76
Q

Which of the following is true about gene therapy?

A

It alters only dysfunctional cells

77
Q

You are a highly paid biotechnologist working for a company that makes the enzyme, amylase, for use in fabric production. Your boss gives you a project that could potentially make the company lots of money. The project is to design a process that uses plasmids and E. coli to make amylase in mass quantities. (Another bacterium will be the source of your gene, but it cannot make enough enzyme compared to recombinant E. coli). Use all of your biotechnology knowledge to come up with a plan. Your job depends on it.

A

Answer: I will need to follow the five basic steps to create a recombinant E. coli.

  • Obtain DNA: Lyse source bacterial cell with amylase gene and collect DNA.
  • Cut DNA at restriction sites: Use a restriction enzyme which makes staggered cuts to cut DNA into multiple fragments of variable lengths with “sticky ends” (one fragment will contain the amylase gene).
  • Insert gene of interest into vector: Use plasmid because it provides origin of replication and ability to jump species. Use the same restriction enzyme to cut plasmid vector open. Amylase gene will hybridize into plasmid.
  • Insert vector into host cell (mass producer): Host cells are E. coli which easily and quickly grow in a lab setting, allowing mass production of the gene. Use artificial transformation (heat shock or electroporation) to introduce recombinant plasmid into E. coli cell, then culture bacteria.
  • Collect product: Lyse E. coli cells and purify amylase.
78
Q

How would the process differ if:

  1. the source of the gene was eukaryotic e.g. human amylase gene?
  2. you used phages as vectors?
  3. you wished to collect the amylase gene, not the amylase enzyme?
  4. you used Bacillus subtilis as a host?
A

The process would differ because:

  1. you would need to create cDNA from mRNA (cDNA has introns removed from the amylase gene).
  2. you would use a phage to infect the E. coli cell with the amylase gene through the process of artificial transduction.
  3. you would confirm the colony containing the amylase gene using colony blotting and DNA probes, then collect the amylase genes by lysing cells.
  4. you would collect the product differently: collect and purify secreted amylase from B. subtilis (no need to lyse cells).
79
Q

What is the first step in cloning a human gene into E. coli?

A

obtain mRNA

80
Q

Restriction enzymes make straight or staggered cuts at:

A

specific DNA sequences

81
Q

Which DNA identification technique will you use to determine all the genes that are expressed in both normal and cancerous human cells?

A

DNA microarray

82
Q

T/F: DNA sequencing is essential for the design of DNA probes.

A

True

83
Q

Which biotechnological method will you use to amplify a gene of interest from a small DNA sample, which you extracted from the fossil remains of an animal?

A

PCR

84
Q

You are a highly paid biotechnologist working for a company that makes vaccines. Your boss gives you a project that could potentially make the company lots of money. The project is to design a process that uses yeast to make a vaccine against herpes simplex virus-2 (a DNA virus). Note: You are looking for something from the herpes simplex virus-2 that creates an immune response (e.g. a protein spike) that you can use to make a vaccine. Also, note that yeast acts similarly to Bacillus subtilis as a host. Use all of your biotechnology knowledge to come up with a plan. Your job depends on it.

A

Answer: I will need to follow the five basic steps to create a recombinant yeast.

  1. Obtain DNA: Lyse source virus with protein spike gene and collect DNA.
    (How would this procedure differ if you started with an RNA virus? What extra step would be needed? Create DNA using reverse transcriptase
  2. Cut DNA at restriction sites: Use a restriction enzyme which makes staggered cuts to cut DNA into multiple fragments of variable lengths with “sticky ends” (one fragment will contain the protein spike gene).
  3. Insert gene of interest into vector: Use plasmid because it provides origin of replication and ability to jump species. Use the same restriction enzyme to cut plasmid vector open. Protein spike gene will hybridize into plasmid.
  4. Insert vector into host cell (mass producer): Host cells are E. coli which easily and quickly grow in a lab setting, allowing mass production of the gene. Use artificial transformation (heat shock or electroporation) to introduce recombinant plasmid into yeast cell, then culture yeast.
  5. Collect product: Collect and purify secreted protein spikes (for vaccine production) from yeast cells.
85
Q

What are restriction enzymes? Explain the two steps involved in RFLP. Give one use of
this technique.

A

Restriction enzymes are enzymes that recognize specific DNA sequences and cut DNA at these sites. recognition sequence is usually 4-6 base, which results in fragments of DNA. The two steps involved in RFLP are cutting the DNA sequences through restriction enzymes and visualizing the fragments using gel electrophoresis.
One use of this technique is to figure out the father of a child, knowing the mother and child’s DNA.

86
Q

what are DNA fragments separated by in gel electrophoresis?

A

size

87
Q

ADD DISCUSSION HERE

A

Microbiology (53 decks)

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