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
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
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 can be put into e.coli
→ It is 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?
→ Human insulin → Interferons → Erythropoietin → Factor XIII → Tissue plasminogen activator
What is the effect of adding control elements to a plasmid?
→ Make genes inducible or express the gene to high levels
What are the requirements for cloning a defective gene to be expressed in large amounts in bacteria?
→ Ability to replicate in bacteria → Maintained at a high copy number → modified origin of replication → selectable (has an antibiotic marker) → Ampicillin resistance gene → Easy to manipulate - cut and rejoin → Multiple cloning site (MCS)
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
What is an inducible promoter?
→ It allows large cultures to be grown without expressing the foreign protein
→ can be turned on and off
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
→ 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
How do you substitute the prokaryotic promoter with a eukaryotic one?
→ Introduce a 3’ UTR containing the polyadenylation signal
→ Terminator must be substituted with a eukaryotic transcriptional terminator
What is an example of a viral promoter?
→ Cytomegalovirus
How do you purify the protein using the epitope tag method?
→ Fuse the recombinant protein with 6 histidines at the 3’ end of the coding sequence
→ histidine is used with nickel affinity columns
→ The histidine binds the protein to the nickel column
→ the purified protein is eluted through
How do you purify the protein using the protein tag method?
→ Add a GST (glutathione-S-transferase) tag at the 3’ end
→ This binds an antibody which is attached to an affinity column
→ This purifies it from bacterial components
How do you localize a protein insert in the cells?
→ You add a green fluorescent protein
→ it is biochemically inert
→ you shine a light on the cells and see where the protein is located within the cells
What is a YAC?
→ Yeast artificial chromosome
How can you alter the properties of a gene product in a plasmid?
→ Can make the gene be secreted extracellularly
→ Fuse gene to a peptide or tag it
