Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Molecular 3rd year > Lecture 4: Plasmids > Flashcards

Lecture 4: Plasmids Flashcards

1
Q

what is genetic transformation

A

the insertion of a gene into an organism

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what are some uses for genetic transformation, and examples

A
  • agriculture genes coding for resistance against frost, pest or spoilage being introduced to plants
  • bioremediation bacteria that can be genetically trannsformed w genes enabling them to digest oil spills
  • medicine diseases caused by defective genes can betrated by gene therapy
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

true/false all vectors are plasmids, but not all plasmids are vectors

A

true

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what is a vector

A

a plasmid that has been manipulated to make it a useful tool

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

what is a plasmid

A
  • small, circular, double stranded, extrachromosomal elements found in some strains of bacteria
  • range from 1 to 200 kb
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what is the copy number of plasmids in a cell

A

from 1-1000

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

how are plasmids normally drawn

A

as a double circle (to represent the 2 DNA strands)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what kind of tension are plasmids under

A

super helical tension (negative supercoiling)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

why are plasmids under so much tension

A

cause they’re closed circular double-stranded DNA molecules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

true/false because all plasmids have the same molecular weight, they migrate at the same rate on an agarose gel

A
  • false
  • different topological forms of plasmid DNA migrate at diff rates
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

rank which different topological forms of plasmid DNA migrate the quickest vs the slowest

A
  • slowest
  • concatamers
  • nicked circular form
  • linear form
  • supercoiled form
  • quickest
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what are the genes encoded on plasmid DNA

A
  • the genes required for its own replication and propagation
  • Origin of replication (ori)
  • Gene for partitioning of plasmids into each daughter cell during cell division
  • Plasmids also often contain genes that confer advantages to their bacterial hosts
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

true/false Genes required for plasmid replication and propagation are stored on plasmid DNA

A

true

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is put in plasmids to create vectors

A
  • Selectable markers that add the thing you want
  • Multiple cloning sites
  • Regulatory signals
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what do selectable markers do when developing cloning vectors

A
  • antibiotic resistant gene
  • allows only bacteria containing plasmid to grow in presence of antibiotic
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what do multiple cloning sites do when developing cloning vectors

A

requires removal and addition of various restriction enzyme sites so a region on the plasmid would contain a number of unique rstriction enzyme sites

17
Q

what do regulatory signals do when developing cloning vectors

A
  • regulate expression of cloned genes
  • promotor sequences to allow expression (transcription) of gene cloned into plasmid
18
Q

what induces the promotor sequence in the pGLO plasmid

A

sugar arabinose

19
Q

what resistance gene is in the pGLO plasmid

A

ampicillin

20
Q

what happens in DNA cloning

A
  • the fragment of DNA contianing the gene of interest is isolated
  • then the fragment of DNA containing the gene is inserted into a plasmid to produce a recombinant DNA molecule
  • introduce the recombinant DNA molecule into a host cell
  • after that, plate the transformed bacteria under selection so that cells containing the plasmid will grow and form a colony
21
Q

what are the diff ways bacteria can receive DNA

A
  • transformation free DNA enters cell
  • transduction DNA containing virus introduces DNA
  • conjugation plasmid in donor cell introduces DNA
22
Q

describe conjugation/ bacterial mating

A
  • a natural plasmid transfer
  • responsible for the spread of antibiotic rsistance in bacteria for viruses
23
Q

what is the most simple way to introduce foreign DNA in the lab

A

CaCl2 treatment and heat shock method

24
Q

describe how genes can be cloned using bacteria

A
  • we start w a circular, double-stranded plasmid DNA (cloning vector)
  • it gets cleaved with a restriction nuclease
  • DNA ligase covalently links a DNA fragment that we want cloned into the vector
  • this plasmid DNA vector is introduced into a bacterial cell
  • the cell culture produced hundreds of Dmillions of new bacteria
25
Q

true/false CaCl2 and heat shock methods are easy and have a high transfer efficiency

A
  • false
  • they are easy but have a low efficiency
26
Q

describe the CaCl2 treatment and heat shock method

A
  • the bacterial cells are incubated with cold solution of CaCl2
  • heat shock the cells to open pores in the cell membrane
  • this allows the foreign DNA to enter
  • then the cells recover and pores close
27
Q

true/false in the CaCl2 treatment and heat shock method, less than one cell will take up the plasmid/vector intended

A

true

28
Q

Why is a good selectable marker important in the CaCl2 treatment and heat shock method?

A

This is because if only 1% of cells take the vector and there is 1000, how do you know which one has the marker, you kill the rest of them (ex: antibiotic resistence)

29
Q

true/false electroporation has high transformation efficiencies

A

true

30
Q

describe the process of electroporation

A
  • Short pulses of current create temporary pores or holes in the cell membrane
  • the DNA can then enter the cell
  • then the cells are allowed to recover
31
Q

what methods can be used for prokaryotic AND eukaryotic transformation

A
  • electroporation
  • lipofection
  • ballistic/ gene gun
32
Q

what is the efficiency of electroporation

A

10^8 to 10^9

33
Q

what happens during lipofection

A
  • DNA is encapsulated in an artificial liposome (micelle)
  • DNA/ liposome complex is added to cells
  • this complex becomes closely associated w the negatively charged cell membrane and eventually fuses w it
34
Q

describe how genes are cloned using bacteria

A
  • start w a circular, double-stranded plasmid DNA cloning vector
  • it is cleaved by a restriction nuclease
  • DNA ligase covalently links a DNA fragment we want to clone into the vector
  • this forms recombinant DNA
  • the recombinant DNA is introduced to a bacterial cell
  • the cell culture produces hundreds of millions of new bacteria
35
Q

what is the deterrent of electroporation

A

slightly more dangerous and more expensive

36
Q

Why is lipofection not commonly used if it is one of the best methods for transformation?

A

Because it is extremely expensive. One liposome can cost anywhere from 10-20$

37
Q

What is Microinjection for DNA transformation?

A

Using a very refined glass micropipette, plasmids are injected into a cell

38
Q

What happens in the Ballistic/gene gun method?

A
  • Gold particles coated with DNA and plasmids is shot at the cell at high velocity
  • This allows stable transformation or transient expression of new genes
39
Q
A

Molecular 3rd year (9 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions