Recombinant DNA And Cloning Vectors

Question 1

What are non-primate lentiviruses used for?

Answer 1

Vectors used to integrate DNA in mammalian cells

Question 2

What are baculoviruses used for?

Answer 2

Vectors used in combination with recombinant expression in insect cells

Question 3

What are artificial chromosomes used for?

Answer 3

→ 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

Question 4

What are plasmids?

Answer 4

→ 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

Question 5

What can plasmids be exchanged between?

Answer 5

Bacteria within a restricted host range

Question 6

What are vectors?

Answer 6

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

Question 7

How are vectors used?

Answer 7

→ 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

Question 8

What are the 6 important features of plasmid vectors?

Answer 8

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

Question 9

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

Answer 9

→ 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

Question 10

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

Answer 10

→ 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

Question 11

Give three reasons why plasmids are used as recombinant tools

Answer 11

→ 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

Question 12

What are 5 recombinant proteins in clinical use?

Answer 12

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

Question 13

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

Answer 13

Make genes inducible or express the gene to high levels

Question 14

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

Answer 14

(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)

Question 15

What control elements are needed for expression in bacteria?

Answer 15

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

Question 16

What is a constituitive promoter?

Answer 16

→ 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

Question 17

What is an inducible promoter?

Answer 17

→ 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

Question 18

Why are constitutive promoters bad?

Answer 18

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

Question 19

What does the inducible promoter typically use?

Answer 19

the lac operator

Question 20

How does the lac operator work?

Answer 20

→ 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

Question 21

How is the lactose operator de-repressed?

Answer 21

Lactose mimic IPTG

Question 22

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

Answer 22

→ 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

Question 23

Why are some proteins expressed in eukaryotes and not prokaryotes?

Answer 23

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

Question 24

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

Answer 24

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

Question 25

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

Answer 25

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

Question 26

What is an example of a viral promoter?

Answer 26

Cytomegalovirus

Question 27

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

Answer 27

→ 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

Question 28

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

Answer 28

→ 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

Question 29

How do you localize a protein insert in the cells?

Answer 29

→ 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

Question 30

How will the transcribed sequence be translated into a protein?

Answer 30

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

Question 31

What are Shine-Dalgarno sequences?

Answer 31

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

Question 32

How is 5’ gene fusion carried out?

Answer 32

→ 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

Question 33

Why are viral promotors used in eukaryotic expression systems?

Answer 33

Because they are more compact and simpler to manipulate

Question 34

Compare eukaryotic and prokaryotic expression vectors

Answer 34

→ 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

Question 35

What do inducible proteins allow?

Answer 35

→ 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

Question 36

How do plasmids alter the properties of the gene product?

Answer 36

→ 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