Brainscape's Knowledge GenomeTM

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


See full index

Genomics > Recombinant DNA and Cloning Vectors > Flashcards

Recombinant DNA and Cloning Vectors Flashcards

1
Q

What are the most commonly used extra-chromosomal genetic elements?

A

Plasmids

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

Where do extra-chromosomal genetic elements originate from?

A

A natural source but are manipulated to use in microbiology

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

What are the recombinant vectors that are used?

A
  • Plasmids
  • Phages
  • Viruses
  • Artifical Chromosomes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Where are plasmids found?

A

In many but not all bacteria

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

Where are phages found?

A

Lambda - bacterial viruses

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are phages used for?

A

They are used as therapeutic tools to treat infectious diseases

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

What are examples of viruses used as recombinant vectors and what are each used for?

A

Non-primate lentiviruses - used to integrate DNA in mammalian cells
Baculoviruses - used in combination with recombinant expression in insect cells

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

What artificial chromosomes are used and what for?

A

Yeast artifical chromosomes - YAC for introducing large segments DNA

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

Why are plasmids essential for recombination?

A
  • Discrete circular dsDNA molecules
  • Means by which genetic information is maintained
  • Genetic elements (replicons) that exist and replicate independently of the bacterial chromosomes so extra-chromosomal
  • Normally exchanged between bacteria within a restricted host range
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are vectors?

A

Cut down version of naturally occuring plasmids

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are vectors used for?

A

Used as molecular tools to manipulate genes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How can a plasmid be modified?

A

It can be used and modified to allow us to introduce a foreign DNA into the plasmid and then it will be maintained as the plasmid replicates within the cell. It can be modified to express a protein or be tagged to look at how a particular protein works within the cell.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are the important features of plasmid vectors?

A
  1. Can be linearised at one or more sites in non-essential stretches of DNA
  2. Can have DNA inserted into them
  3. Can be re-circularised without loss of the ability to replicate.
  4. Modified to replicate at high multiplicity (copy number) within a host cell
  5. Contain selectable markers
  6. Are relatively small 4-5kb in size
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Where are restriction sites found in the bacterial plasmid DNA?

A

Found at the portion that is “non-essential”

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is formed in the plasmid and what does this do?

A

A cloning site is introduced to the plasmid and this will introduce restriction enzymes as it will contain multiple restriction sites.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How are recombinant proteins made from recombinant DNA?

A

The transduce bacteria is where the plasmids will replicate and be maintained. This will isolate that which will express the recombinant gene. This can be used to produce recombinant proteins in bacteria.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What are recombinant proteins needed for?

A

To investigate their properties

To develop and produce therapeutics

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Why are plasmids used as a recombinant tools?

A
  • Expression of a recombinant gene in a living organism of choice
  • Add or modify control elements
  • Alter the properties of the gene product
  • Make it useful as a therapeutic
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How are recombinant proteins used clinically?

A

Recombinant proteins or peptides constitue about 30% of all biopharmaceuticals.

  • Human insulin
  • Interferons
  • Erythryopoietin
  • Factor XIII
  • Tissue plasminogen activator (TPA)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What is an increasingly important drug class?

A

Recombinant antibodies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What are the requirements for a plasmid in the prokaryotic system?

A
  • Ability to replicate in bacteria
  • Maintained at high copy number
  • Selectable contains an antibiotic marker
  • Easy to manipulate
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What control elements are required for expression in bacteria?

A
  • The coding sequence
  • Shine-Dalgarno sequence
  • Promoter
  • Transcriptional terminator
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What is the coding sequence?

A

The part of the gene coding for the protein not including the UTRs nor any intronic or regulatory sequences such as a promoter enhancers.

24
Q

What is the shine-dalgarno sequence?

A

The ribosomal binding site found around 8 nucleotides before the start codon in the RNA in prokaryotes.

25
Q

What is the promoter?

A

The gene element that is involved in regulation and initiation of transcription.

26
Q

What is the transcriptional terminator?

A

A sequence that terminates transcription and initiates the dissociation of transcription.

27
Q

What is a constitutive promoter?

A

Promoter that is always on.

28
Q

What is a constitutive promoter used for?

A

It allows a culture of cells to express the foreign proteins to a high level.

29
Q

When should a constitutive promoter not be used?

A

It is fine if the protein isn’t toxic to E.coli. It is a bad idea if there is no cells that would be able to survive and produce a recombinant protein.

30
Q

What is a inducible promoter?

A

It is a molecular switch

31
Q

What is a inducible promoter used for?

A

It allows large cultures to be grown without expressing the foreign protein. It induces a response to a defined signal.

32
Q

What do inducible promoters use?

A

Transcriptional repressors

33
Q

What is lac Operator?

A

It is used by a inducible promoter that is de-repressed by addition of lactose mimic IPTG.

34
Q

Describe structure of lac operon

A

Comprises of genetic elements that in prokaryotes include some regulatory sequences one of which is the lac operator and a gene that lac repressor (inhibitor).

35
Q

What does the lac operon system allow?

A

It allows bacteria to be responsive to low glucose environments and switch to lactose as a carbon source.

36
Q

How is any gene regulated by the lac operon?

A

This system to regulate any gene by placing a lac operator (lacO) upstream of its transcriptional start.

37
Q

Requirements for gene regulation

A
  • Copy of the coding sequence
  • Must contain the start codon to and including the stop codon
  • Easy to manipulate
38
Q

Why are some proteins best made in eukaryotes?

A
  • Many pharmacologically useful proteins are heavily modified and will not be appropriately processed in bacteria.
  • Solution is to express them in a eukaryotic system
39
Q

How is the effect of a defective gene in a cell culture system studied?

A
  • Express the protein in human embryonic fibroblasts
40
Q

What is used to study the effect of a defensive gene?

A
  • Inducible Promoter
  • Shine-Delgarno
  • Insert with in frame start and stop codon
  • Transcriptional terminator
  • Origin of replication
  • Selectable marker
  • Choice of unique Restriction sites MCS
41
Q

What problem can be encountered when studying for a defective gene?

A
  • Bacterial promoter doesn’t work
  • Shine-Delgarno sequence isn’t recognised
  • Transcriptional start is not recognised, no cap site
  • No polyadenylation signal
  • Termination of transcription not recognised by eukaryotic Polymerase II
  • Origin of replication doesn’t work
42
Q

Compare prokaryotic and eukaryotic expression vectors

A

Prokaryotic:

  • Contains a promoter
  • Shine-dalgarno sequence
  • ORF (common in humans rare in E.coli - codon preference)
  • Terminator

Eukaryotic:

  • Promoter
  • Kozak sequence
  • ORF
  • Introns can be tolerated but aren’t necessary
  • Poly-A signal in 3’ UTR
  • Terminator
43
Q

What happens to plasmids?

A

They transfect into the eukaryotic system

44
Q

How are plasmids grown up in bacteria?

A
  • Selectable bacterial marker

- Maintained at high copy number

45
Q

What are the changes that occur in a prokaryotic vector?

A
  • Substiution of promoter with a eukaryotic promoter
  • Introduce a 3’ UTR containing polyadenylation signal
  • Terminator substituted with a eukaryotic transcriptional terminator
46
Q

What is a transgenic cell line (transient or stable expression)?

A
  • Ability to replicate mammalian cells or integrated in the chromosomes
  • A selectable marker in eukaryotes is needed
47
Q

Describe the structure of the eukaryotic/prokaryotic hybrid vector

A

Contains:

  • Eukaryotic promoter RSV, CMV
  • Choice of unique Restriction sites MCS with Kozack consensus
  • Transcriptional terminator and polyadenylation signal BGH
  • 2nd Eukaryotic promoter SV40
  • Eukaryotic selectable marker
  • Transcriptional terminator and polyadenylation signal SV40
  • Bacterial origin of replication
  • Bacterial selectable marker ampR
48
Q

Why are viral promoters commonly used?

A

They are used in eukaryotic expression systems because they are more compact and simpler to manipulate

49
Q

What is the combined transcriptional terminator and polyadenylation signal used?

A

3’UTR of Bovine Growth Hormone (BGH)

50
Q

When are selectable markers used and give an example of one

A

G418, one of many, but must be constructed with eukaryotic promoter polyadenylation signal and terminator and SV40 virus sequences

51
Q

Why can functional analysis of a protein not occur?

A

The expressed protein was very hard to obtain in a pure enough form from the bacteria

52
Q

What are the two of the most popular 3’ gene fusions protein tags?

A

6 Histidines and Glutathione S transferase (GST)

53
Q

Where are 3’ gene fusions found?

A

Made at either end of the coding sequence either before the stop codon or after the start

54
Q

Explain the steps of forming recombinant proteins from recombinant DNA

A
  • Insert recombinant plasmid DNA into the recombinant expression vector
  • Transform into E.coli
  • Forms colonies on agar plate containing antibiotic
  • Isolate colonies; confirm insert and culture
  • Induce and purify protein
  • Purify protein on affinity column (separates tagged protein)
  • Elute
55
Q

How is the localisation and trafficking of the protein in a cell culture system studied?

A

By expressing the protein in human embryonic fibroblasts

56
Q

What needs to be figured out when studing a protein in a human cell?

A

Where it goes in cells - cytoplasmic, nucleus or in a membrane

57
Q

How was the fate of a protein tracked?

A
  • In 1971, a fluorescent protein was identified and cloned from Jelly Fish.
  • The green colour derives from an intrinsically green fluorescent protein, that is non-toxic and otherwise biochemically inert.
  • 5’ gene fusions are used and a protein tag is added to track the fate of a protein.

Genomics (19 decks)

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