Recombinant DNA and cloning vectors

Question 1

What recombinant vectors are there in the molecular tool kit?

Answer 1

-Plasmids
-Phages
-Lambda(They’re bacterial viruses)
-Viruses
-Non-primate lentiviruses
-Vectors used to integrate DNA in mammalian cells
-Baculoviruses
-Vectors used in combination with recombinant
expression in insect cells
-Artificial chromosomes
-Yeast artificial chromosome
-Introducing large segments of DNA

Question 2

What are plasmids?

Answer 2

Plasmids are discrete circular dsDNA molecules found in many but not all bacteria

Question 3

Why are plasmids extrachromosomal?

Answer 3

Because they’re genetic elements that exist and replicate independently of the bacterial chromosomes and and therefore extra chromosomal

Question 4

What can plasmids normally be exchanged between?

Answer 4

Can normally be exchanged between bacteria within a restricted host range

Question 5

What are vectors?

Answer 5

Vectors are a cut down version of naturally occuring plasmids and are used as molecular tools to manipulate genes

Question 6

What are the characteristics of a plasmid as a vectors?

Answer 6

1. Can be linearized at one or more sites in non-essential stretches of DNA
2. Can have DNA inserted into them
3. Can 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. Are relatively small, 4-5 kbs in size

Question 7

Why do we use plasmids as recombinant tools?

Answer 7

-Plasmids add functionality over simple DNA and facilitate functional genomics
-Expression of a recombinant gene in a living organism of
choice
-Add or modify control elements
-Alter the properties of the gene product
-Make it useful as a therapeutic

Question 8

What are biologics?

Answer 8

Biologics are recombinant antibodies

Question 9

What are the requirements for a plasmid in a prokaryotic system?

Answer 9

• Ability to replicate in bacteria
• Maintained at high copy number
  
  • Modified origin of replication
• Selectable contains an antibiotic marker
  
  • ampR gene
• Easy to manipulate- cut and re-join
• Multiple cloning sites

Question 10

What are the control elements required for expression in bacteria?

Answer 10

A gene coding sequence with:

• A shine dalgarno sequence for ribosome binding site recognition of AUG
• Bacterial promoter
• Transcriptional terminator

Question 11

What 2 things can a promoter be?

Answer 11

Promoter can be constitutive or inducible

Question 12

What does it mean if the promoter is constitutive?

Answer 12

• Always on
• Allows a culture of cells to express the foreign protein to a high level
• Fine if the protein isn’t toxic to E-coli

Question 13

What does it mean if the promoter is inducible?

Answer 13

• Molecular switch
• Allows large cultures to be grown without expressing the foreign protein
• Induced in response to a defined ligand

Question 14

What do inducible promoters use?

Answer 14

1. Use transcriptional repressors

2. Use lac operator which is de-repressed by addition of lactose mimic IPTG

Question 15

What is the comparison between eukaryotic and prokaryotic expression vectors?

Answer 15

• The shine dalgarno sequence in prokaryote is substituted for a kozac sequence in eukaryote
• Poly A tail in a 3’ UTR added in eukaryotic just before eukaryotic terminator

Question 16

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

Answer 16

1. Vector that’s easy to manipulate- cut and rejoin
2. Can also be grown up in bacteria:
   - Selectable bacterial marker
   - Maintained at high copy number
3. Substitution of a promoter with a eukaryotic promoter
4. Introduce a 3’ UTR containing poly-A signal
5. Terminator must be substituted with eukaryotic transcriptional terminator