Recombinant DNA and Cloning Vectors Flashcards
We can use plasmids as vectors; however, they aren’t the only molecular tool used as such.
List some examples of other recombinant vectors.
PHAGES:
- lambda: bacterial viruses
VIRUSES:
- non-primitive Lentiviruses: 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 YACs: introducing large segments of DNA
Define plasmids.
They are discrete circular dsDNA molecules found in many, but not all bacteria.
They are a means by which genetic information is maintained in bacteria.
They are genetic elements (replicons) that exist and replicate independently of the bacterial chromosome, and are therefore extra-chromosomal.
They can normally be exchanged between bacteria within a restricted host range (eg. plasmid-borne antibiotic resistance).
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
(prokaryote or eukaryote) - add or modify control elements
(make it inducible or express it to high levels on demand) - alter the properties of the gene product
(make it secreted extra-cellularly or into the periplasmic space)
(fuse it to a peptide tag or other protein) - make it useful as a therapeutic
Describe recombinant proteins in clinical use.
Recombinant vectors facilitate the production of recombinant drugs.
Recombinant proteins or peptides constitute about 30% of all biopharmaceuticals, such as:
- human insulin - diabetes
- interferons α and β - viral hepatitis or MS
- erythropoietin - kidney disease, anaemia
- Factor XIII - haemophilia
- tissue plasminogen factor (TPA) - embolism, stroke
Around 62 recombinant drugs were approved by the FDA for clinical use between 2011 and 2016.
Describe recombinant antibodies (biologics) in clinical use.
- they first appeared in the clinic in the late 1980s
- made up around 50% of recombinant drugs approved by the FDA between 2011 and 2016
- 17 biologics were approved for 2017, double the previous average
- have an estimated 18.5 billion dollars of sales in the US in 2010
- they are becoming increasingly important as a drug class
What are some advantages when using a plasmid in a prokaryotic system?
- ability to replicate in bacteria (eg. E. Coli)
- has a modified origin of replication to be maintained at a high copy number
- contains a selectable antibiotic marker (such an ampicillin resistance gene)
- easy to manipulate - can cut and rejoin (multiple cloning sites, MCS)
What control elements are required for expression in bacteria?
- gene coding sequence
- Shine-Dalgarno sequence (-8) RBS recognition of AUG
- bacterial promoter
- transcriptional terminator
All ‘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
- [bad idea if it is]
INDUCIBLE - molecular switch
- allows large cultures to be grown without expressing the foreign protein
- induced in response to a defined signal
Inducible promoters use transcriptional repressors.
As a recap, describe the use of the lac operon.
The lac operon comprises genetic elements that, in prokaryotes, induce some regulatory sequences, one of which is the lac operator and a gene, the lac repressor (inhibitor). 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 (lacO) upstream of its transcriptional start.
Some proteins are best made in eukaryotes.
Expand.
Many pharmocologically useful proteins are heavily modified (post-transcriptionally) and will not be appropriately processed in bacteria. Examples include interferons.
Usually, this modification is by glycosylation. Some proteins retain biological activity, some don’t.
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 will the requirements change for a plasmid being transfected into a eukaryotic system, as compared to a prokaryotic system?
- we still need a vector that is easy to manipulate (we can cut and rejoin it)
- it can also be grown up in bacteria:
- have a selectable bacterial marker
- maintained at a high copy number
- substitution of the promoter with a eukaryotic promoter
- introduce a 3’ UTR containing a polyadenylation signal
- the terminator must be substituted with a eukaryotic transcriptional terminator
- it must have transient or stable expression (ie. a transgenic cell line)
- with the ability to replicate mammalian cells
- or integrated into the chromosomes
- for this, 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 on to it, which can then be used to purify it (to then perform a functional analysis of the protein).
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, but two of the most popular are 6 Histidines and Glutathione S Transferase (GST).
Describe 5’ gene fusions.
In this case, we add the GFP tag after the start codon, and use it to track the fate of the protein.
GFP stands for Green Fluorescent Protein. The green colour derives from an intrinsically green fluorescent protein that is non-toxic and otherwise biochemically inert.
