Recombinant DNA And Cloning Vectors**

Question 1

Give examples of various recombinant vectors in molecular biology

Answer 1

• Plasmids
• Bacteriophages
• Viruses
• Artificial Chromosomes

Question 2

Describe PLASMIDS as an example of a recombinant vector in molecular biology

Answer 2

• Found in many but not all bacteria
• Generally have a restricted host range
• Are transferable by various means including transformation and conjugation

Question 3

Describe Bacteriophages as an example of a recombinant vector in molecular biology

Answer 3

• Lambda - Bacterial viruses
• Transfer of anti microbial resistance through a mechanism called transduction

Question 4

Describe VIRUSES as an example of a recombinant vector in molecular biology

Answer 4

• Non primate Lentiviruses - Vectors used to integrate DNA into mammalian cells
• Baculoviruses - Vectors used in combination with recombinant expression in insect cells

Question 5

Describe ARTIFICIAL CHROMOSOMES as an example of a recombinant vector in molecular biology

Answer 5

Yeast artificial chromosomes - introducing large segments of DNA

Question 6

How are Plasmids an essential part of the molecular tool kit?

Answer 6

They’re:
- Discrete Circular dsDNA molecules found in many but not all bacteria
- Are a means by which genetic information is maintained in bacteria
- Are genetic elements that exist and replicate independently of the bacterial chromosomes and are therefore extra chromosomal
- Can normally be exchanged between bacteria within a restricted host range (e.g plasmid borne antibiotic resistance)

Question 7

Describe what a vector in DNA is

Answer 7

A cut down version of naturally occurring Plasmids & are used as molecular tools to Manipulate genes

Question 8

What are the features of Plasmid Vectors? (VD)

Answer 8

• Can be linearised at one or more sites in non essential stretches of DNA
• Can have DNA inserted into them
• Can be re-circularised without loss of ability to replicate
• Are often modified to replicate at high multiplicity within a host cell
• Contains selectable markers
• Most are relatively small 4-5kb in size

Question 9

Describe bacterial plasmids as vectors

Answer 9

View diagram

Question 10

Describe recombinant proteins from recombinant DNA

Answer 10

View diagram

Question 11

Why should we use plasmids as recombinant tools?

Answer 11

Plasmids add functionality over simple DNA and facilitate experimental or functional genomics such as:

• Expression of a recombinant gene in a living organism of choice: prokaryote or Eukaryote
• Add or modify control elements: Make it inducible or express it to high levels on demand
• Alter the properties of the gene products: Make it secreted extra cellularly or into the periplasmic space, or fuse is to a peptide tag or other protein
• Make it useful as a therapeutic

Question 12

Describe the clinical use of recombinant proteins

Answer 12

Recombinant vectors facilitate the production of recombinant drugs

Recombinant proteins or peptides constitute about 30% of all bio pharmaceuticals such as:
- Human insulin: diabetes
- Interferons a & b: Viral Hepatitis or MS
- Erythropoietin: Kidney disease, anaemia
- Factor XIII: Haemophilia
- Tissue plasminogen activator (TPA): Embolism, stroke

Question 13

SCENARIO QUESTION:
I want to clone the defective gene from a patient with an inherited condition and express it in bacteria in large amounts so that I can perform functional analysis on the protein?

Question 14

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

Answer 14

• Ability to replicate in bacteria
• Maintained at a high copy number
• Modified origin of replication
• Selectable contains an antibiotic marker
• Ampicillin resistance gene
• Easy to manipulate - Cut and Rejoin
• Multiple cloning sites

Question 15

What control elements are required for expression in Bacteria?

Answer 15

• Gene coding sequence insufficient
• Shine dalgarno sequence (-8) RBS recognition of AUG (still not enough)
• Bacterial promoter (still not enough)
• Transcriptional terminator

Question 16

What is a constitutive promoter?

Answer 16

• Constitutive: Always on
• Allow a culture of cells to express the foreign protein to a high level
• Fine if the protein isn’t toxic to E. coli

Question 17

What is an Inducible Promoter?

Answer 17

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

Question 18

Which is preferred more? Constitutive or Inducible

Answer 18

Constitutive Promoters

Question 19

What type of Repressors do inducible promoters use?

Answer 19

• Inducible Promoters typically uses the lac operator which de-repressed by addition of lactose mimic called IPTG
• Requires the constitutive expression of lac inhibitor

Question 20

What are the requirements for the DNA inserts?

Answer 20

• DNA must be easy to manipulate
• Copy of the coding sequence
• Must contain the start codon to & the stop codon
• No introns
• No Cap site required
• No eukaryotic UTRs required
• No Polyadenylation signal required

Question 21

Where are some proteins best made in? Eukaryotes or Prokaryotes

Answer 21

Eukaryotes
- Many pharmacological useful proteins are heavily modified and will not be processed in bacteria. eg interferons
- Some proteins retain biological activity but others don’t
- The solution is to express them in a eukaryotic system