9. Recombinant DNA and Cloning Vectors Flashcards
What is used for the production of recombinant vectors and how are these used?
• PLASMIDS - not in all bacteria, but they are involved in the spreading of antibiotic resistance from one organism to another in the context of a hospital infection.
•PHAGES. e.g. lambda
•VIRUSES:
- NON-PRIMATE LENTIVIRUSES - vectors used to integrate DNA in mammalian cells
- BACULOVIRUSES - vectors used in combination with recombinant expression in insect cells ~ shows expression in a eukaryotic system (in insect and not in mammalian cell, for safety)
•ARTIFICIAL CHROMOSOMES - Yeast artificial chromosomes (YACs) – introducing large segments DNA
Why are virsues limited to the size of DNA molecule they can carry.
Because they need a capsid for their DNA material.
•The larger the size of DNA they need to carry, the more unstable it is less they are likely to maintain. - This is why YACs are useful (they are in the eukaryotic system)
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
- Are genetic elements (replicons) that exist and replicate independently of the bacterial chromosomes and are therefore extra-chromosomal
- Can normally be exchanged between bacteria within a restricted host range (e.g. plasmid borne antibiotic resistance) - cannot ‘jump’ from one organism to another, they have to be compatible.
What are vectors?
Vectors are a cut down version of naturally occurring Plasmids & are used as molecular tools to Manipulate genes
What are the important features of plasmid vectors?
1) Can be linearized at one or more sites in non-essential stretches of DNA
- non-essential stretches as bases may be lost or inserted, which could lead to loss of function if in essential stretches.
2) Can have DNA inserted into them
3) And can be re-circularised without loss of the ability to replicate
4) Are often modified to replicate at high multiplicity (copy number) within a host cell
5) Contain selectable markers - e.g. antibiotics like ampicilin or tetracyclin
6) Are relatively small 4-5kb in size
How is plasmid DNA linearized?
It is cut by a single enzyme – you can insert a piece of DNA in that.
Linearise the plasmid so you can have the right restriction sites on the linear DNA.
How is directional insertion of the PCR DNA product achieved?
- Within the primer sequence generated, there are restriction enzyme sites at the end that have been modified so they can then be put by restriction enzymes.
- There is different restriction enzymes at each site - allow directional insertion of the PCR product.
How do obtain recombinant proteins from recombinant DNA?
1) Insert DNA and form recombinant plasmid DNA
2) Transform the recombinant plasmid DNA into E.coli
3) Transfer the E.coli to agar plate containing antibiotic
4) Only populations with the antibiotic resistance (in the recombinant DNA) will form colonies and the rest will die off
5) You can isolate a colony and confirm the insert by restriction mapping, or PCR
6) You then isolate colonies and culture them
7) Then you purify the proteins - (you can first add the mimic IPTG if the promoter is inducible to induce the production of proteins)
Why use plasmids as recombinant tools?
Plasmids add functionality over simple DNA and facilitate functional genomics:
1) Expression of a recombinant gene in a living organism of choice prokaryote or eukaryote
2) Add or modify control elements - make it inducible or express it to high levels on demand
3) 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
4) Make it useful as a therapeutic
Examples of recombinant proteins in clinical use
• Recombinant proteins or peptides constitute about 30% of all biopharmaceuticals
- Human insulin - diabetes - Interferons-α & β – viral - Hepatitis or MS - Erythropoietin – kidney disease, anaemia - Factor VIII – haemophilia - Tissue plasminogen activator (TPA) – embolism, stroke - Around 50% of the 62 recombinant drugs approved by the FDA for clinical use between 2011 and 2016 are biologics (antibodies) ~ increasingly important as a drug class
What are requirements from a plasmid in a prokaryotic system?
- Ability to replicate in bacteria (E. coli)
- Maintained at high copy number - modified origin of replication
- Selectable contains an antibiotic marker - Ampicillin resistance gene
- Easy to manipulate – cut and re-join - choice of unique restriction sites multiple cloning site (MCS) to linearise it in a way you choose.
What control elements are required for expression in bacteria?
1) Shine-Dalgarno sequence (– 8) RBS recognition of AUG (this is the ribosomal binding site)
2) Bacterial promoter
3) Transcriptional terminator
4) Gene coding sequence - intons should be spliced out beforehand
What is the difference between a constitutive promoter and an inducible promoter?
•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 - use transcriptional repressors (typically uses lac Operator which is de-repressed by addition of lactose mimic IPTG)
RECAP: Briefly describe the lac operon
- lac Operator (lacO) is upstream of its transcriptional start - This is a binding site near the promoter, and when bound by a protein Lac inhibitor (LacI) it prevents the transcription of the gene (as it is in the way of the initiating polymerase complex)
- In the presence of lactose (when glucose in the environment is too low to be used a glucose source) – lactose binds to the LacI and this de-represses the LacO and so the promotor - and LacI dissociates from the LacO
- Dissociation of LacI from the LacO allows transcription of the gene
- We don’t use lactose in this process – we use a mimic of it called IPTG.
Requirements of a DNA sequence.
•Copy of the coding sequence – e.g. by PCR
•Must contain the start codon to & including the stop codon
- No introns – bacteria can’t splice it – ie exonic sequence only
- No Cap site required
- No eukaryotic UTRs required
- No polyadenylation signal required – bacterial RNAs are not polyadenylated
- Shine-Dalgarno seqeunce
•Easy to manipulate – cut and re-join to other DNA add restriction sites using PCR or other methods
