Recombinant DNA and cloning vectors Flashcards
What are some examples of recombinant vectors?
PHAGES:
- lambda: bacterial viruses
VIRUSES:
- non-primitive Lentiviuses: vectors used to integrate DNA in mammalian cells
- Baculoviruses: vectors used in combination with recombinant expression in insect cells (a eukaryotic system)
ARTIFICIAL CHROMOSOME:
- yeast artificial chromosomes: introducing large segments of DNA
Define plasmids
Discrete circular dsDNA molecules found in many bacteria. They are a means by which genetic information is maintained in bacteria
They are genetic elements (replicons) that exist and replicate independelty of the bacterial chromosome, and are therefore extra-chromosomal
They can be exchanged between bacteria within a restricted host-range
What are the characteristics of a plasmid?
- can be linearised at one or more sites in non-essential stretches of DNA
- can have DNA inserted into them
- can be circularised without loss of the ability to replicate
- are often modified to replicate at high multiplicity (copy number) within a host cell
- contain selectable markers
- are relatively small, 4-5kb in size, making them easy to manipulate
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
- add or modify control elements
- alter the properties of the gene product (make it secreted extra-cellularly)
- make it useful as a therapeutic
Describe the use of recombinant proteins in clinical use
They facilitate the production of recombinant drugs, like
- human insulin
- interferons a and b
- viral hepatitis
- erythropoietin
- factor XIII
- tissue plasminogen factor
Advantages of using a plasmid in a prokaryotic system?
- ability to replicate in bacteria
- has a modified origin of replication to be maintained at a high copy number
- contains a selectable antibiotic marker
- easy to manipulate
- can cut and rejoin
What control elements are required for expression in bacteria?
- gene coding sequence
- shine-dalgarno sequence
- bacterial promoter
- transcriptional terminator
all of these parts are needed for successful expression
Describe constitutive and inducible promoters
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
INDUCIBLE:
molecular switch
- allows large cultures to be grown without expressing the foreign protein
- induced in response to a defined signal
Recap the use of the lac operon
Comprises genetic elements that induce some regulatory sequences, one of which is the lac operator and a gene, the lac repressor. These allow bacteria to be responsive to low glucose environments and switch to lactose as a carbon source.
We can use this system to regulate any gene by placing a lac operator upstream of its transcriptional start
Why do we use a eukaryotic system to express proteins?
Many pharmacologically useful proteins are heavily modified and will not be appropriately processed in bacteria, e.g. interferons.
Usually, this modification is caused by glycosylation. Some proteins retain biological activity, some don’t. So, the solution is to express them in a eukaryotic system
Compare what is required for prokaryotic and eukaryotic vectors?
PROKARYOTIC:
- prokaryotic promoter
- Shine-Dalgarno sequence
- ORD (codon preference)
- prokaryotic terminator
EUKARYOTIC:
- enhancer
- eukaryotic promoter
- Kozak sequence
- ORF
- eukaryotic terminator
How are the requirements different for a plasmid being transfected into a eukaryotic system, as compared to a prokaryotic system?
- we need a vector that is easy to manipulate
- it must have a selectable bacterial marker
- it is maintained at a high copy number
- the promoter must be substituted with a eukaryotic promoter
- there must be a 3’ UTR containing a polyadenylation signal
- the terminator must be substituted with a eukaryotic transcriptional terminator
- it must have transient/stable expression with the ability to replicate mammalian cells/be integrated into the chromosomes
- we need a selectable marker in eukaryotes
Describe 3’ gene fusions
The aim of this is to make a protein which has additional sequences/amino acids added onto it, which can then be used to purify it
Fusions can be made at either end of the coding sequence, either before the stop codon or after the start
Many different protein tags are used, with the two most popular being 6 Histidines and GST
Describe 5’ gene fusions
In this case, we add the GFP (green fluorescent protein) tag after the start of the codon, and use it to track the fate of the protein. The green colour derives from an intrinsically fluorescent protein that is non-toxic and biochemically inert
