Recombinant DNA and cloning vectors Flashcards
What recombinant vectors are there in the molecular tool kit?
- PLASMIDS found in many but not all bacteria (most commonly used)
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PHAGES
- Lambda – bacterial viruses
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VIRUSES
- Non-primate Lentiviruses –vectors used to integrate DNA in mammalian cells
- Baculoviruses –vectors used in combination with recombinant expression in insect cells (a eukaryotic expression system)
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ARTIFICIAL CHROMOSOMES
- Yeast artificial chromosomes (YACs) are extra chromosomal and replicate independently, maintains foreign DNA – introducing large segments DNA
What are plasmids?
Plasmids are discrete circular dsDNA molecules found in many but not all bacteria
Genetic elements (replicons) which exist and replicate independantly of the bacterial chromosome = extra-chromosomal
Why are plasmids extrachromosomal?
Because they’re genetic elements that exist and replicate independently of the bacterial chromosomes and and therefore extra chromosomal
What can plasmids normally be exchanged between?
Can normally be exchanged between bacteria within a restricted host range
What are vectors?
Vectors are a cut down version of naturally occuring plasmids
- used as molecular tools to manipulate genes
What are the characteristics of a plasmid as a vectors?
- Can be linearized at one or more sites in non-essential stretches of DNA
- Can have DNA inserted into them
- Can re-circularised without loss of the ability to replicate
- Are often modified to replicate at high multiplicity within a host cell
- Contain selectable markers e.g AB resistance
- Are relatively small, 4-5 kbs in size
Why do we use plasmids as recombinant tools?
Plasmids add functionality over simple DNA and facilitate functional genomics
- Expression of a recombinant gene in a living organism of choice (prokaryote/eukaryote)
- Add or modify control elements (make it inducible or express to high levels on demand)
- Alter the properties of the gene product (make it secreted extracellularly or into periplasmic space)
- Make it useful as a therapeutic
What are biologics?
Biologics are recombinant antibodies
What are advantages of using a plasmid in a prokaryotic system?
- -Ability to replicate in bacteria
- -Maintained at high copy number
- -Modified origin of replication
- -Contains selectable antibiotic marker (e.g ampicillin resistance gene)
- -Easy to manipulate= can cut and re-join (Multiple cloning sites, MCS)
What are the control elements required for expression in bacteria?
- A gene coding sequence with:
- A shine dalgarno sequence for ribosome binding site recognition of AUG
- Bacterial promoter
- Transcriptional terminator
What 2 things can a promoter be?
Promoter can be constitutive or inducible
What does it mean if the promoter is constitutive?
- 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 (bad idea if it is)
What does it mean if the promoter is inducible?
- Molecular switch
- Allows large cultures to be grown without expressing the foreign protein
- Induced in response to a defined signal (e.g can allow the expression of the protein to be stopped before it causes E.coli to be toxic)
What do inducible promoters use?
Give an example of this
They use transcriptional repressors
E.g Use lac operator which is de-repressed by addition of lactose mimic IPTG
- LacO is the operator and LacI will produce a protein
- LacI protein will bind to the operator preventing transcription from the promoter
- When you want transcription to occur you will cause derepression of the promoter by adding IPTG
What is the comparison between eukaryotic and prokaryotic expression vectors?
- The shine dalgarno sequence in prokaryote is substituted for a kozac sequence in eukaryote
- Prokaryotic terminator is substituted for a eukaryotic terminator
- PolyA tail and 3’UTR s added to eukaroyote
- Enhancer also added to eukaryote
What are the requirements for plasmids transfected into a eukaryotic system?
- Vector that’s easy to manipulate- cut and rejoin
- Can also be grown up in bacteria: -Selectable bacterial marker -Maintained at high copy number
- Substitution of a promoter with a eukaryotic promoter
- Introduce a 3’ UTR containing poly-A signal
- Terminator must be substituted with eukaryotic transcriptional terminator
What are 3’ gene fusions?
- Makes a protein which has additional sequences/amino acids added onto it which are then used to purify it
- Fusions can be made at either end of the coding sequence either before the stop codon or after the start
e.g 6His tag binds to nickel if passed through a nickel column and binds the protein selectively to the column, everything else is washed through resulting in the pure protein
What are 5’ gene fusions?
Used to track the fate of a protein, has it gone to the cytoplasm,nucleus or membrane?
- A GFP (green fluorescent protein) is added after the start codon and is used to track the fate of the protein
