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

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

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 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

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

→ Human insulin 
→ Interferons 
→ Erythropoietin 
→ Factor XIII 
→ Tissue plasminogen activator

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 requirements for cloning a defective gene to be expressed in large amounts in bacteria?

Answer 14

→ 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)

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

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

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

Question 30

What is a YAC?

Answer 30

→ Yeast artificial chromosome

Question 31

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

Answer 31

→ Can make the gene be secreted extracellularly

→ Fuse gene to a peptide or tag it