Recombinant DNA And Cloning Vectors Flashcards
What are non-primate lentiviruses used for?
Vectors used to integrate DNA in mammalian cells
What are baculoviruses used for?
Vectors used in combination with recombinant expression in insect cells
What are artificial chromosomes used for?
→ Introducing large segments of DNA
→ Used because large pieces of DNA are unstable and unlikely to be incorporated into plasmids
→ Similar conceptually to plasmids but are much bigger and are restricted to yeast
What are plasmids?
→ Discrete circular dsDNA molecules found in many but not all bacteria
→ Are a means by which genetic information is maintained in bacteria
→ Genetic elements (replicons) that exist and are replicated independently of the bacterial chromosomes
What can plasmids be exchanged between?
Bacteria within a restricted host range
What are vectors?
A piece of DNA that is circular and foreign DNA can be inserted within this
How are vectors used?
→ The plasmid is cut so the ends of the plasmid are complementary with the PCR product
→ Piece of DNA can be ligated
→ Used to mutate a gene and understanding the functional role of parts of a protein or the effects of a specific mutation on protein structure or function
→ To insert promoters in front of reporter genes allowing us to better understand the regulatory mechanisms of a gene’s promoter
What are the 6 important features of plasmid vectors?
1) they can be linearised at one or more sites in non-essential stretches of DNA
2) can have DNA inserted into them
3) can be 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) relatively small in size
What are the steps to use a bacterial plasmid as a vector?
→ Linearise it at a particular restriction site
→ Generate a PCR product of the gene you want which is then restricted
→ Include within the primer sequence of the gene a restriction enzyme site
→ Plasmid is restricted to allow insertion of a DNA product
→ Gene is then ligated
How do you select the plasmids that have taken up the gene?
→ The plasmid put into e.coli
→ Then plated onto agar containing antibiotic that corresponds to the antibiotic resistance gene that has been inserted
→ Only the plasmids that contain the gene will grow and form colonies
→ The colony can then be cultured and isolated
→ Confirm insertion by restriction mapping a clone
Give three reasons why plasmids are used as recombinant tools
→ Plasmids can express a recombinant gene in a living organism of choice
→ you can add or modify control elements
→ alter properties of the gene product
What are 5 recombinant proteins in clinical use?
1) Human insulin- diabetes
2) Interferons(alpha and beta)- viral hepatitis or MS
3) Erythropoietin- kidney disease, anaemia
4) Factor XIII- haemophilia
5) Tissue plasminogen activator- embolism, stroke
What is the effect of adding control elements to a plasmid?
Make genes inducible or express the gene to high levels
What are the 7 requirements for cloning a defective gene to be expressed in large amounts in bacteria?
(1) → Ability to replicate in bacteria
(2) → Ampicillin resistance gene
(3) → Easy to manipulate - cut and rejoin
(4) → Maintained at a high copy number
(5) → Modified origin of replication
(6) → Multiple cloning site (MCS)
(7) → Selectable (has an antibiotic marker)
What control elements are needed for expression in bacteria?
→ Shine dalgarno sequence (ribosomal binding site for prokaryotes)
→ Bacterial promoter
→ Transcriptional terminator
What is a constituitive promoter?
→ It is always on
→ Allows a culture of cells to express the foreign protein to a high level which is fine if the E.coli is toxic
What is an inducible promoter?
→ It allows large cultures to be grown without expressing the foreign protein
→ can be turned on and off
→ induced in response to a defined signal
Why are constitutive promoters bad?
→ If the protein is toxic to E.Coli
→ the sequence will kill the bacteria
What does the inducible promoter typically use?
the lac operator
How does the lac operator work?
→ When lactose is absent the repressor binds to the operator
→It prevents RNA polymerase from binding to the promoter
→ When lactose is present and the enzymes are needed
→ Lactose binds to the repressor protein
→ this changes the shape of the repressor
→ It can no longer bind to the operator
→ RNA polymerase can bind to the promoter and the enzyme is transcribed
How is the lactose operator de-repressed?
Lactose mimic IPTG
What are the requirements for a eukaryotic gene to be used in a bacterial plasmid?
→ must contain the start codon and include the stop codon
→ no introns- bacteria can’t splice it
→ no cap site
→ no eukaryotic UTRs
→ no polyadenylation signal is required - bacterial RNAs are not polyadenylated
Why are some proteins expressed in eukaryotes and not prokaryotes?
→ Proteins are heavily modified and cannot be processed in bacteria eg interferons by glycosylation
→ Some proteins retain biological activity and some don’t
What are the requirements for a plasmid transfected into a eukaryotic system?
→ Eukaryotic promoter
→ Kozak sequence (Shine-Dalgarno isn’t recognized)
→ Cap site
→ Polyadenylation signal - eukaryotic terminator
