8. RECOMBINANT DNA & CLONING Flashcards
What are the 4 main recombinant vectors used?
- Plasmids
- Viruses
- Bacteriophages
- Artificial Chromosomes
What are plasmids?
- Plasmids are discrete, circular double stranded molecules found in many but not all bacteria
- Plasmids can exist & replicate independently of the chromosome so they are extra-chromosomal
- Plasmids have a restricted host range
- Plasmids are a way of storing genetic material, it can be transferred via transformation or conjugation (sex pilli)
***What are bacteriophages?
- Bacteriophages are viruses that infect bacteria, they are known as Lamda
- Viruses can transfer their genetic material into bacteria & fuse the bacteria to produce multiple viruses
- The process by which viruses transfer genetic info from one bacterium to another is called TRANSDUCTION
What viruses are used to express recombinant DNA?
- Non-primate lentiviruses are used to insert DNA into mammalian cells
- Baculoviruses are used to insert DNA into a eukaryotic system such as insects
Where are the artificial chromosomes for recombinant technology taken from?
- The artificial chromosomes are taken from yeast. Known as YAC - Yeast Artificial Chromosome
**What are 6 features of plasmid vectors?
- Plasmid vectors are relatively small (4-5Kb)
- Plasmid vectors can be linearized at multiple sites in non-essential stretches of DNA
- Plasmid vectors can have DNA inserted into them
- Plasmid vectors can be modified to have high copy number
- Plasmid vectors can be recircularised without losing the ability to replicate
- Plasmids contain selectable markers (antibiotic resistance markers)
**How are recombinant proteins produced from recombinant DNA?
- Recombinant DNA transformed into E.Coli
- Placed on an agar plate containing antibiotic
- Recombinant plasmids should produce colonies
- Colonies can be isolated & cultured
- Protein can then be purified
**Why are plasmids used as recombinant tools?
- Plasmids can increase the functionality of a gene
1. Expression of a recombinant gene
2. Add or modify control elements
3. Alter the properties of genes
Give examples of 5 recombinant proteins for clinical use?
- Factor XIII - Hemophilia
- Insulin - Type 1 Diabetes
- Interferon - Viral Hepatitis
- Erythropoietin - Kidney disease & Anaemia
- Tissue Plasminogen Factor - Stroke & Embolism
What are the requirements for cloning a gene in a bacteria?
- Must be able to replicate in bacteria
- Must be easy to replicate
- Must have a high copy number
- Must have a modified origin of replication
- Must be easy to manipulate
- Multiple cloning sites
What 3 control elements are required for expression in bacteria?
- SHINE-DALGARNO SEQUENCE - a sequence of 8 bases upstream from AUG
- TRANSCRIPTION TERMINATOR
- BACTERIAL PROMOTER
What are the two types of promoters?
- Constitutive
2. Inducible
What is a constitutive promoter?
- A CONSTITUTIVE PROMOTER is always on, so it can express proteins to a high level
- However, this can be damaging if the protein is toxic
- It is also energetically expensive
*What is a inducible promoter?
- An INDUCIBLE PROMOTER acts as a molecular switch
- They allow large cultures to be grown, without expressing the recombinant protein
- Even if the protein is toxic, it will have very little effect on the culture growth
- Inducible promoters use transcriptional repressors which can bind & dissociate
What are the requirements of a DNA insert that is to be placed in a prokaryotic system?
- DNA must be easy to manipulate
- Must be a copy of the coding sequence
- Must include a start & stop codon
- Must not have introns as bacteria can’t splice
- Shouldn’t have a cap site, polyadenylation signal or eukaryotic UTRss
Why should the DNA insert for a prokaryotic system not have introns?
- Prokaryotes cannot splice RNA and so they do not recognise RNA splice sites in eukaryotic genes and prokaryotic genes do not therefore have introns.
- The sequence should be exonic & contain no introns, cap site, polyadenylation or eukaryotic UTRs
- It is important that any sequence included in the inserted plasmid vector corresponds to the final coding sequence of the mature RNA.
*Why can’t a bacterial gene be put into a eukaryotic cell?
- A shine-dalgarno sequence won’t be recognised
- Bacterial promoters don’t work in eukaryotes
- Transcriptional termination & start site isn’t recognised
- No cap site or polyadenylation
- Origin of replication doesn’t work
*What are the differences between eukaryotic & prokaryotic vectors?
PROKARYOTIC
- Shine dalgarno sequence
- Prokaryotic promoter & transcriptional terminator
EUKARYOTIC
- Has an enhancer
- Eukaryotic promoter
- Cap site/ Kozak sequence instead of Shine -Dalgarno
- Eukaryotic terminator (stop codon) & Poly A tail
How can a bacterial vector be modified to be inserted into a eukaryotic system?
- Introduce 3’UTR
containing Polyadenylation signal - Substitute prokaryotic promoter for eukaryotic promoter
- Substitute prokaryotic terminator for a eukaryotic terminator (stop codon)
How can a recombinant protein be made easier to purify?
- The colonies from the agar plate can be isolated & cultured
- The recombinant protein can be purified with an AFFINITY COLUMN
- An affinity column separates the tagged protein
How can the localisation & trafficking of proteins be studied?
- GFP - Green florescent protein is a fusion tag which can be used to track protein location
- GFP is non-toxic, absorbs light at 395nm & emits light at 509nm
