Recombinant DNA + cloning vectors

Question 1

Give examples of various recombinant vectors in molecular biology:
Plasmids
Bacteriophages
Viruses
Artificial Chromosomes

Answer 1

• Plasmids: most commonly used ,
  restrictive host range
  transfer genetic material via transduction
  most non essential genes are already cut from plasmid so small (5kb bases) can be implemented
• Bacteriophages: strict range due to instability of large DNA segments
  can conjugate to other bacteria
• Viruses: used commonly for reverse transcription into host cells to encourage production of desired product
• Artificial Chromosomes: YAC’s typically used to introduce large segments (whole gene)

Question 2

Describe in detail the features of plasmids that make them useful for manipulating fragments of DNA and expression of recombinant proteins, with examples

Answer 2

Features:
- exist and replicate independent of bacteria
- conjugative plasmids are not transferred horizontally (antibiotic resistance)
- used to understand interactions between gene products in biological system (yeast=>hybrid)
- flexibility (linearised and recirculised)
- modulation of expression (modified to produce high copy number replication)
- contain selectable markers

Question 3

Describe the use of inducible promoters in plasmids

Answer 3

constitutive: gene remains switched on
- cell culture can express protein to high level
- inappropriate if protein in non-toxic to e.coli
inducible: induced response to specific/defined signal
- large cultures can be formed w/out expression/ production of protein
- reduces proteins toxicity to bacteria

Question 4

Describe the relevant differences between prokaryotic and eukaryotic gene expression requirements

Answer 4

Prokaryotic: - select markers (eg: antibiotic resistance/ amipicillin)
- maintain high copy number
- replicate in bacteria (e.coli)
- multiple cloning sites
- restriction sites (easy manipulated DNA insert)
- shine-dalgarno
- transcription terminator
- lac operator/repressor (low glucose environments switch to lactose)

Eukaryoates:
- bacteria promoters unrecognised
- shine dalgarno unrecognised (requires kozac sequence)
- transcriptional start, polyadenylation signal (or has to be in 3’UTR), cap site nor transcriptional terminator is NOT recognised by eu.RNA Pol 2
- Stable expression or transient?

Question 5

Describe the use of gene fusions to improve purity of recombinant proteins

Answer 5

3’ gel fusions: making proteins easier to purify
- 6 histodines
- GST tag
- has to be placed in correct order
- causes high affinity binding of HisTAG
= separation of protein from others = easier purification

GFP
- absorbance 395nm
- 5’ gel fusion
- must be placed after AUG codon
- fluorescent microscopy allows tracking of the protein