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πšπšŽπš—πš˜πš–πš’πšŒπšœ > Recombinant DNA and Cloning Vectors > Flashcards

Recombinant DNA and Cloning Vectors Flashcards

1
Q

What are non-primate lentiviruses used for?

A

β†’ vectors used to integrate DNA in mammalian cells

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2
Q

What are baculoviruses used for?

A

β†’ vectors used in combination with recombinant expression in insect cells

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3
Q

What are artificial chromosomes used for?

A

β†’ introducing large segments of DNA

β†’ 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

β†’ Discrete circular dsDNA molecules found in many but not all bacteria
β†’ Are a means by which genetic information is maintained in bacteria
β†’ 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

β†’ bacteria within a restricted host range

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6
Q

What are vectors?

A

β†’ 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

β†’ The plasmid is cut so the ends of the plasmid are complementary with the PCR product
β†’ 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

β†’ Linearise it at a particular restriction site
β†’ generate a PCR product of the gene you want which is then restricted
β†’ Include within the primer sequence of the gene a restriction enzyme site
β†’ plasmid is restricted to allow insertion of a DNA product
β†’ gene is then ligated

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10
Q

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

A

β†’ The plasmid can be put into e.coli
β†’ It is then plated onto agar containing antibiotic that corresponds to the antibiotic resistance gene that has been inserted
β†’ only the plasmids that contain the gene will grow and form colonies
β†’ The colony can then be cultured and isolated
β†’ confirm insertion by restriction mapping a clone

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11
Q

Give three reasons why plasmids are used as recombinant tools?

A

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

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12
Q

What are 5 recombinant proteins in clinical use?

A 
β†’ Human insulin 
β†’ Interferons 
β†’ Erythropoietin 
β†’ Factor XIII 
β†’ Tissue plasminogen activator
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13
Q

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

A

β†’ 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 
β†’ Ability to replicate in bacteria 
β†’ Maintained at a high copy number 
β†’ modified origin of replication
β†’ selectable (has an antibiotic marker) 
β†’ Ampicillin resistance gene 
β†’ Easy to manipulate - cut and rejoin 
β†’ Multiple cloning site (MCS)
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15
Q

What control elements are needed for expression in bacteria?

A

β†’ Shine dalgarno sequence (ribosomal binding site for prokaryotes)
β†’ Bacterial promoter
β†’ Transcriptional terminator

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16
Q

What is a constituitive promoter?

A

β†’ It is always on

β†’ Allows a culture of cells to express the foreign protein to a high level

17
Q

What is an inducible promoter?

A

β†’ It allows large cultures to be grown without expressing the foreign protein
β†’ can be turned on and off

18
Q

Why are constitutive promoters bad?

A

β†’ If the protein is toxic to E.Coli

β†’ the sequence will kill the bacteria

19
Q

What does the inducible promoter typically use?

A

β†’ the lac operator

20
Q

How does the lac operator work?

A

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

21
Q

How is the lactose operator de-repressed?

A

β†’ Lactose mimic IPTG

22
Q

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

A

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

23
Q

Why are some proteins expressed in eukaryotes and not prokaryotes?

A

β†’ Proteins are heavily modified and cannot be processed in bacteria
β†’ Some proteins retain biological activity and some don’t

24
Q

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

A

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

25
Q

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

A

β†’ Introduce a 3’ UTR containing the polyadenylation signal

β†’ Terminator must be substituted with a eukaryotic transcriptional terminator

26
Q

What is an example of a viral promoter?

A

β†’ Cytomegalovirus

27
Q

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

A

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

β†’ histidine is used with nickel affinity columns
β†’ The histidine binds the protein to the nickel column
β†’ the purified protein is eluted through

28
Q

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

A

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

29
Q

How do you localize a protein insert in the cells?

A

β†’ You add a green fluorescent protein
β†’ it is biochemically inert
β†’ you shine a light on the cells and see where the protein is located within the cells

30
Q

What is a YAC?

A

β†’ Yeast artificial chromosome

31
Q

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

A

β†’ Can make the gene be secreted extracellularly

β†’ Fuse gene to a peptide or tag it

πšπšŽπš—πš˜πš–πš’πšŒπšœ (19 decks)

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