Recombinant DNA And Cloning Vectors Flashcards

1
Q

What are non-primate lentiviruses used for?

A

Vectors used to integrate DNA in mammalian cells

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2
Q

What are baculoviruses used for?

A

Vectors used in combination with recombinant expression in insect cells

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3
Q

What are artificial chromosomes used for?

A

→ Introducing large segments of DNA
→ Used because large pieces of DNA are unstable and unlikely to be incorporated into plasmids
→ Similar conceptually to plasmids but are much bigger and are restricted to yeast

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4
Q

What are plasmids?

A

→ Discrete circular dsDNA molecules found in many but not all bacteria
→ Are a means by which genetic information is maintained in bacteria
→ Genetic elements (replicons) that exist and are replicated independently of the bacterial chromosomes

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5
Q

What can plasmids be exchanged between?

A

Bacteria within a restricted host range

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6
Q

What are vectors?

A

A piece of DNA that is circular and foreign DNA can be inserted within this

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7
Q

How are vectors used?

A

→ The plasmid is cut so the ends of the plasmid are complementary with the PCR product
→ Piece of DNA can be ligated
→ Used to mutate a gene and understanding the functional role of parts of a protein or the effects of a specific mutation on protein structure or function
→ To insert promoters in front of reporter genes allowing us to better understand the regulatory mechanisms of a gene’s promoter

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8
Q

What are the 6 important features of plasmid vectors?

A

1) they 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) are often modified to replicate at high multiplicity within a host cell
5) contain selectable markers
6) relatively small in size

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9
Q

What are the steps to use a bacterial plasmid as a vector?

A

→ Linearise it at a particular restriction site

→ Generate a PCR product of the gene you want which is then restricted

→ Include within the primer sequence of the gene a restriction enzyme site

→ Plasmid is restricted to allow insertion of a DNA product

→ Gene is then ligated

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10
Q

How do you select the plasmids that have taken up the gene?

A

→ The plasmid put into e.coli
→ Then plated onto agar containing antibiotic that corresponds to the antibiotic resistance gene that has been inserted
→ Only the plasmids that contain the gene will grow and form colonies
→ The colony can then be cultured and isolated
→ Confirm insertion by restriction mapping a clone

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11
Q

Give three reasons why plasmids are used as recombinant tools

A

→ Plasmids can express a recombinant gene in a living organism of choice
→ you can add or modify control elements
→ alter properties of the gene product

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12
Q

What are 5 recombinant proteins in clinical use?

A

1) Human insulin- diabetes
2) Interferons(alpha and beta)- viral hepatitis or MS
3) Erythropoietin- kidney disease, anaemia
4) Factor XIII- haemophilia
5) Tissue plasminogen activator- embolism, stroke

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13
Q

What is the effect of adding control elements to a plasmid?

A

Make genes inducible or express the gene to high levels

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14
Q

What are the 7 requirements for cloning a defective gene to be expressed in large amounts in bacteria?

A

(1) → Ability to replicate in bacteria
(2) → Ampicillin resistance gene
(3) → Easy to manipulate - cut and rejoin
(4) → Maintained at a high copy number
(5) → Modified origin of replication
(6) → Multiple cloning site (MCS)
(7) → Selectable (has an antibiotic marker)

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15
Q

What control elements are needed for expression in bacteria?

A

→ Shine dalgarno sequence (ribosomal binding site for prokaryotes)
→ Bacterial promoter
→ Transcriptional terminator

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16
Q

What is a constituitive promoter?

A

→ It is always on
→ Allows a culture of cells to express the foreign protein to a high level which is fine if the E.coli is toxic

17
Q

What is an inducible promoter?

A

→ It allows large cultures to be grown without expressing the foreign protein
→ can be turned on and off
→ induced in response to a defined signal

18
Q

Why are constitutive promoters bad?

A

→ If the protein is toxic to E.Coli
→ the sequence will kill the bacteria

19
Q

What does the inducible promoter typically use?

A

the lac operator

20
Q

How does the lac operator work?

A

→ When lactose is absent the repressor binds to the operator
→It prevents RNA polymerase from binding to the promoter
→ When lactose is present and the enzymes are needed
→ Lactose binds to the repressor protein
→ this changes the shape of the repressor
→ It can no longer bind to the operator
→ RNA polymerase can bind to the promoter and the enzyme is transcribed

21
Q

How is the lactose operator de-repressed?

A

Lactose mimic IPTG

22
Q

What are the requirements for a eukaryotic gene to be used in a bacterial plasmid?

A

→ must contain the start codon and include the stop codon
→ no introns- bacteria can’t splice it
→ no cap site
→ no eukaryotic UTRs
→ no polyadenylation signal is required - bacterial RNAs are not polyadenylated

23
Q

Why are some proteins expressed in eukaryotes and not prokaryotes?

A

→ Proteins are heavily modified and cannot be processed in bacteria eg interferons by glycosylation
→ Some proteins retain biological activity and some don’t

24
Q

What are the requirements for a plasmid transfected into a eukaryotic system?

A

→ Eukaryotic promoter
→ Kozak sequence (Shine-Dalgarno isn’t recognized)
→ Cap site
→ Polyadenylation signal - eukaryotic terminator

25
Q

How do you substitute the prokaryotic promoter with a eukaryotic one?

A

→ Introduce a 3’ UTR containing the polyadenylation signal
→ Terminator must be substituted with a eukaryotic transcriptional terminator

26
Q

What is an example of a viral promoter?

A

Cytomegalovirus

27
Q

How do you purify the protein using the epitope tag method?

A

→ Fuse the recombinant protein with 6 histidines at the 3’ end of the coding sequence

→ Histidine is used with nickel affinity columns

→ Histidine binds the protein to the nickel column

→ The purified protein is eluted through

28
Q

How do you purify the protein using the protein tag method?

A

→ Add a GST (glutathione-S-transferase) tag at the 3’ end
→ This binds an antibody which is attached to an affinity column
→ This purifies it from bacterial components

29
Q

How do you localize a protein insert in the cells?

A

→ You add a green fluorescent protein which is biochemically inert
→ You shine a light on the cells and see where the protein is located within the cells

30
Q

How will the transcribed sequence be translated into a protein?

A

→ If the sequence contains the correct in frame start and stop codons,
→ Upstream of which there should ideally also be a Shine Dalgarno sequence.

31
Q

What are Shine-Dalgarno sequences?

A

A ribosomal binding site in bacterial and archaeal messenger RNA, generally located around 8 bases upstream of the start codon AUG

32
Q

How is 5’ gene fusion carried out?

A

→ Relies upon the insertion of the GFP coding sequence (minus the stop codon) either immediately before the stop or after the start codon.

→ The following gene must remain in the same reading frame for the correct decoding of its sequence so that the chimeric protein is correctly translated

33
Q

Why are viral promotors used in eukaryotic expression systems?

A

Because they are more compact and simpler to manipulate

34
Q

Compare eukaryotic and prokaryotic expression vectors

A

→ Bacterial codon usage has a different frequency than that of eukaryotes. For example the arginine codon AGG is common in humans but rare in bacteria.

→ The Shine-Dalgarno sequence is replaced by capping

→ The identification of the correct start codon is partly defined by the Kozak sequence in a 5’ UTR

→ Introns are tolerated but are not necessary

→ A polyadenylation signal is required in a 3’ UTR

35
Q

What do inducible proteins allow?

A

→ Large cultures to be grown without expressing the foreign protein
→ The production of a protein that is toxic then has little affect on the growth of the culture

36
Q

How do plasmids alter the properties of the gene product?

A

→ Make it secreted extra-cellularly or into the periplasmic space,
→ Fuse it to a peptide tag or other protein
→ Make it useful as a therapeutic