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Biomedical Lab > Lab 6 > Flashcards

Lab 6 Flashcards

1
Q

Competent e.coli bacterial cells are prepared using….

A

Calcium chloride

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2
Q

The competent E.coli are transformed with the _____ ______ ___ using ______

A

PGLO plasmid DNA using heat shock

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3
Q

Non transformed AND transformed E.coli are grown on similar…? What does this detect?

A

Grown on similar agar plates. To detect the phenotypic differences

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4
Q

The E.coli colonies on the various agar plates will be evaluated for what 2 things?

A

Antibiotic resistance and fluorescent phenotypes

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5
Q

What 2 things are isolated and tested to understand the relationship between genotype, gene regulation, and phenotype?

A

PGLO plasmid DNA and GFP (Green fluorescent protein)

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6
Q

Each student will use PGLO transformed E.coli culture grown using ____ or ____

A

Antibiotic selection or Antibiotic selection AND AraC gene regulation

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7
Q

What are prokaryotes?

A

Organisms made up of cells that lack a cell nucleus or any membrane-encased organelles

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8
Q

Give an example of a prokaryote.

A

Bacteria

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9
Q

What are PLASMIDS?

A

Small, circular extrachromosomal DNA found in bacteria

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10
Q

Plasmids replicate _____ of the chromosome and generally result in ______ changes required for survival in a hostile environment

A

Plasmids regulate INDEPENDENTLY of the chromosomes and generally result in PHENOTYPIC changes required for survival in a hostile environment

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11
Q

Give an example of when a plasmid would replicate in attempt to survive in a hostile environment

A

Antibiotic resistance

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12
Q

Plasmid DNA transfer occurs_____ in bacteria through a process known as _____

A

Occurs naturally, transformation

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13
Q

Transformation is used in……

A

Recombinant DNA technology

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14
Q

Can transformation be performed in a lab?

A

YES easily

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15
Q

What is the genome?

A

Complete set of genetic information in an organism

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16
Q

How is the genome stored?

A

In the form of long DNA molecules called chromosomes

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17
Q

What are genes?

A

Short segments of DNA containing the protein coding information.

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18
Q

Where are genes located (don’t say nucleus)

A

In the chromosomal DNA

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19
Q

In higher organisms, where else are genes present besides chromosomal DNA?

A

Mitochondrial DNA

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20
Q

DNA stands for…..

A

Deoxyribonucleic acid

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21
Q

DNA molecules are very long chains of repeated subunits known as…

A

Nucleotides or bases (A,G,C,T)

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22
Q

DNA strands are…

A

Antiparallel

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23
Q

The 2 DNA strands are twisted to form a ——

A

Helix

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24
Q

DNA is also present in which 2 places?

A

In eukaryotic mitochondria and in bacterial plasmids

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25
Mitochondrial and plasma DNA are called…?
Extrachromosomal DNA
26
The antiparallel strand reads __to___
3 prime-5 prime
27
The parallel strand reads
5 prime-3prime
28
DNA is said to be____ed
Supercoiled
29
PGLO contains several ___ and ____ that enable ____
Genes and DNA sequences that enable replication of the plasmid DNA
30
What is GFP?
The jellyfish gene that encodes green fluorescent protein
31
What is responsible for the green fluorescent phenotype?
GFP gene
32
What is ori?
The ORIGIN of pGLO plasmid DNA replication
33
What is ampicillin?
An antibiotic that kills bacteria
34
How is ampicillin destroyed?
By the enzyme beta-lactamase (encoded by bla gene)
35
Do TRANSFORMED BACTERIA die in the presence of ampicillin?
NO-THEY SURVIVE
36
What is bla?
A gene that encodes the enzyme beta-lactamase. Responsible for antibiotic resistant phenotype.
37
Does bla allow bacteria to grow in the presence of ampicillin?
Yes
38
What does the enzyme beta lactamase do and what gene encodes it?
Breaks down ampicillin. Encoded by bla gene
39
What is the pBAD promoter?
Specific DNA sequence upstream from GFP gene. Binds araC-arabinose and promotes RNA polymerase binding and transcription of GFP
40
What does the araC gene encode?
Encodes the regulatory protein that binds to the pBAD promoter
41
When is the production of GFP switched on?
ONLY when arabinose binds to the araC protein
42
What binds to the pBAD promoter?
The araC protein
43
The araC protein is encoded by…
The araC gene
44
What is arabinose?
A sugar that binds to the pBAD promoter and displaces the AraC protein from it so RNA polymerase can bind and initiate transcription
45
How is recombinant DNA formed?
DNA from 1 organism foreign to the bacteria (or yeast) is inserted into their plasmid
46
What is a DNA VECTOR?
The recombinant plasmid formed from inserting a foreign piece of DNA into bacteria
47
The recombinant plasmid is transformed in…?
Cells
48
Recombinant plasmid is transformed in cells. The cells are grown in media to produce more ____ or _____
Plasmids or the gene product (proteins)
49
Recombinant DNA technology is useful in..?
Preparing proteins that are used in research and therapy
50
Recombinant DNA technology involves 4 steps:
1. Synthesis of recombinant plasmid 2. Transformation of the organism 3. Selection of the transformed bacteria 4. Synthesizing the gene product (protein of interest)
51
“Synthesizing a recombinant plasmid” What MUST the plasmid contain?
-Gene of interest (duh) -DNA for replication (origin of replication) -antibiotic resistance gene for survival in an antibiotic containing media
52
What is an optional thing for the plasmid to contain?
Other genes for regulating the transcription of the proteins
53
“Transformation of the organism” What does this mean?
The plasmid is inserted in a bacterial cell
54
“Selection of the transformed bacteria..” What does this mean?
Transformed cell is grown in an antibiotic containing media. Therefore, only the bacterial cells containing the plasmid will grow. Non transformed cells will die
55
“Synthesizing the gene product(protein)” How is this done?
The cells are grown in media that contain molecules required for producing the protein
56
What does it mean to be a competent cell?
The cells are capable of taking in foreign DNA
57
What are cells treated with to make them competent?
Calcium chloride (CaCl2)
58
How does calcium chloride enable cells to be competent?
The positive charge of calcium shields the negative charge of DNA phosphates. Makes it easy for DNA to enter the cell through the cell membrane
59
How are competent cells stored before they are used?
They are frozen
60
While competent cells are kept frozen until use, what prevents the formation of ice crystals and prevents them from dying?
Glycerol
61
Transformation is a method to introduce ____ into _____
Introduce plasmid DNA into bacteria
62
DNA is very ____ charged
Negatively
63
After the bacterial cells uptake the foreign DNA, then what?
Bacteria are shocked using hot/cold treatment. Ice slows fluidity of the membrane. Then, the transformation mixture is quickly warmed up. Shocks the cells and increases permeability of the cell membranes
64
How are shocked cells recovered?
Kept in nutrient broth for a short period of time. Recovered cell suspension is grown overnight on agar plates
65
Each colony represents…
A single transformed cell (clone)
66
Plates without antibiotic are used as…
Controls to demonstrate that the cells were alive and survived the transformation
67
Agar plates WITH antibiotic are used to…
Select the transformed cells. These cells are the antibiotic resistant phenotype and grow in the presence of the antibiotic
68
What is RNase? To what buffer is it added and why?
Enzyme that rapidly degrades RNA. RNASE is added to P1 buffer to exclude the RNA in our DNA extract
69
BUFFER P2 solution that appears clear shows the ____ of cells and _____ of cellular components
lysis, solubilization
70
the cell suspension will turn ___after the addition of P2
blue
71
BUFFER P1 solution that appears cloudy shows the ____of cells
suspension
72
which buffer is the neutralization buffer?
N3
73
in N3, solution changes from blue to…
colorless
74
in N3, the solution appears as a milky precipitate. why?
insoluble DNA and proteins

Biomedical Lab (10 decks)

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