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

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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

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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 can be put into e.coli
→ It is 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
→ Human insulin 
→ Interferons 
→ Erythropoietin 
→ Factor XIII 
→ Tissue plasminogen activator
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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 requirements for cloning a defective gene to be expressed in large amounts in bacteria?

A
→ Ability to replicate in bacteria 
→ Maintained at a high copy number 
→ modified origin of replication
→ selectable (has an antibiotic marker) 
→ Ampicillin resistance gene 
→ Easy to manipulate - cut and rejoin 
→ Multiple cloning site (MCS)
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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

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

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
→ 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
→ The 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
→ it is biochemically inert
→ you shine a light on the cells and see where the protein is located within the cells

30
Q

What is a YAC?

A

→ Yeast artificial chromosome

31
Q

How can you alter the properties of a gene product in a plasmid?

A

→ Can make the gene be secreted extracellularly

→ Fuse gene to a peptide or tag it