Recombinant DNA & cloning vectors Flashcards
What is a vector?
A recombinant tool that can be used to transfer DNA into a biological system like a bacterium or a cell. There are various types of extrachromosomal DNA tools (that we refer to as vectors) that are used in molecular biology.
What four commonly used categories do vectors fall into?
Plasmids, Bacteriophages, Viruses, Artificial chromosomes.
What are bacteriophages/phages?
Lambda is one of the best known phages. They can be thought as a type of bacterial virus.
They are naturally occurring but can also transfer antimicrobial resistance through a mechanism called transduction.
Phages also have restricted host range, their restriction and ability to lyse bacteria means they have been used as antibacterial agents.
What are (recombinant) viruses?
Recombinant viruses are used widely as vectors in eukaryotic systems including animals or even recombinant vaccines.
Lentiviruses are non-primate viral vectors used to integrate DNA in mammalian cells.
Baculoviruses are vectors used in combination with recombinant expression in insect cells. #9a eukaryotic expression system).
What are artificial chromosomes?
Yeast artificial chromosomes (YACs). They are very large DNA molecules used for introducing large segments of DNA for example entire genes including promoters and introns. YACS are similar to plasmids but are much bigger and are restricted to yeast.
What are plasmids?
Most common form of recombinant vector. They are discrete circular double stranded DNA molecules that are only found in prokaryotes (found in some bacteria, but not all), but are used for introducing DNA into both prokaryotes and eukaryotes. They are a source of antimicrobial resistance in the environment.
- They are a means by which genetic info is maintained in bacteria
- They are genetic elements, referred to as replicons, that exist and replicate independently of the bacterial chromosomes and are therefore extra-chromosomal.
- They can normally be exchanged between bacteria by a restricted host range. (eg plasmid born antibiotic resistance.
- They are transferable by various means including transformation and conjugation.
What is the difference between transformation and conjugation?
Conjugation- In the environment, plasmids may be transferred to other bacteria by conjugation. This is where living bacteria form sex pili encoded by the tra genes of a conjugative plasmid.
Transformation- Its also possible for plasmids to be transferred by natural transformation, but this is rare due to the instability of large molecules of DNA in the environment. Experimentally, transformation is the main means by which plasmids are artificially transferred into bacteria.
Commonly used vectors are a cut down version of naturally occurring plasmids. What are they used for?
They are used as molecular tools to manipulate genes. E.g:
- For cloning a gene or disease causing variant of a gene and producing a recombinant protein in a biological system, e,g, bacteria, in large quantities
- used to mutate a gene and understanding the role of parts of a protein or the effects of specific mutation on protein structure or function.
- Used to insert promoters in front of reporter genes allowing us to understand the regulatory mechanisms of a genes promoter.
- Sometimes used in two component systems to understand the interaction and association of different gene products in a biological system for example the yeast two hybrid system.
What are the features of plasmid vectors?
- Can be linearised at one or more sites in non-essential (to replication) stretches of DNA.
- Can have DNA inserted into them – these regions can have DNA inserted without the loss of the ability to replicate or be maintained.
- Can be re-circularised without the loss of ability to replicate
- Often modified to replicate at high multiplicity (copy number) within a host cell. Although not essential its advantageous if plasmids replicate at a high copy number within the bacterium as it allows genes to be expressed to higher levels and the recovery of greater amounts of DNA and gene products for experimental use.
- Contain selectable markers - e.g ampicillin for antibiotic resistance
- Most are relatively small in size (4-5kb) - partly because other non-essential genetic features have been removed.
How can you use bacterial plasmids as a vector? (Brief method)
The selection of the vector is important- needs to have correct features for inserting the gene selecting for recombinants. For example, Vector must have appropriate cassete into which the gene will be inserted. The vector may have a bacterial promotor, a multiple cloning site with a variety of restriction sites where you can cut and linearise it, and a bacterial transcriptional terminator.
The vector and the PCR amplicon of the gene must be cut with restriction enzymes to produce compatible ends. These are then joined together by ligation via DNA ligase.
Should then have a re-circularised recombinant vector containing the gene sequence.
Using the recombinant plasmid made, how do you then make recombinant proteins?
Having made the recombinant plasmid, you can then artificially transduce bacteria where the plasmids will replicate and be maintained in the presence of a selectable marker such as ampicillin.
Can then pick individual clones, grow these up in bulk to produce recombinant proteins in the bacteria where you can then choose to purify the protein produced and investigate its properties/function or alternatively develop and produce therapeutics.
Why use plasmids as recombinant tools?
Plasmids add functionality over simple DNA and facilitate experimental or 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 of a bacterium. Both these make it easier to purify (important if using in therapeutics)
- Fuse it to a peptide tag or other protein
- Make it useful as a therapeutic.
What are some example of recombinant proteins currently used as drugs?
Human insulin- diabetes
Interferons (alpha & beta)- viral hepatitis or MS
Erythropoietin – kidney disease and anaemia
Factor XIII- haemophilia
Tissue plasminogen activator (TPA) - used to treat clots formed as result of embolisms or strokes.
What are the requirements for a plasmid in a prokaryotic system?
- Ability to replicate in bacteria (e.g E.coli)
- Maintained at high copy number , modified replication of origin
- Selectable, contains an antibiotic marker. Should have this to allow us to select transformants e.g ampicillin resistance gene.
- Easy to manipulate – so we can cut and insert large segments of DNA (corresponding to the coding sequence of the gene and have same restriction sites added its ends) and rejoin. Plasmid needs to thus have range of restriction sites within Multiple cloning sites (MCS) that also don’t occur within the genes coding sequence.
What control elements are required for expression in bacteria?
What is necessary for the transcription and translation for the recombinant gene:
- In bacterium, we don’t want the UTRS nor any of the intronic or regulatory sequences, such as promoters and enhancers, parts of the coding sequence. For example, introns can’t be processed in prokaryotic and eukaryotic regulatory sequences won’t work. Hence gene coding sequence is insufficient
- Shine-Dalgarno sequence - the ribosomal binding site found around 8 nucleotides before the start codon in prokaryotes. RBS recognition of AUG. As the RNA of prokaryotes isn’t capped, having the shine-Dalgarno sequence would benefit translational efficiency.
- Bacterial promoter – need a strong bacterial promoter to initiate transcription as this needs to be added to the 5’ end of the transcription unit.
- Transcriptional terminator – to complete the transcription unit, a transcriptional terminator is required to allow the polymerase to end transcription and release the message.
