Recombinant DNA and cloning vectors Flashcards
What is a vector?
Extra-chromosomal genetic elements that are used to artificially carry foreign genetic material into another cell where it can then be replicated and/or expressed
What are the different types of vector?
- Plasmids
- Phages - bacterial viruses
- Viruses - Lentiviruses
- Artificial chromosomes - e.g. yeast artificial chromosomes (YACs)
What is the advantage that artificial chromosomes have over the 3 other types of vector?
Artificial chromosomes are able to carry and introduce large segements of DNA into a cell unlike the other 3 types of vector
What are some of the characteristics of a plasmid?
- Circular double stranded DNA molecules found in bacteria
- Naturally occuring - replicate within the bacteria
- A means by which genetic information is maintained in bacteria
- Extra-chromosomal
- Can normally be exchanged between bacteria
What is meant when it is said that plasmids are “extra-chromosomal?”
It means that plasmids exist and replicate independently from the bacterial chromosomes
Explain what is meant when it’s said that a plasmid can be transferred vertically or horizontally
- Transferred vertically - Transferred from parent to daughter cells of replicating bacteria
- Transferred horizontally - Transferred between compatible bacterial cells via conjugation
What are some of the important features of a plasmid vector?
- Can be linearized in non-essential regions of DNA - This allows for foreign DNA to be inserted into plasmid
- Can have DNA inserted into them
- Can be re-circularised without loss of the ability to replicate
- Can replicate at a high copy number within a host cell
- Must contain selectable markers - e.g. antibiotic resistance markers
- Must be relatively small
Why is it important that a plasmid vector is able to replicate at a high copy number within a cell?
Because the more copies you have of a particular gene within a cell the more that gene will be expressed. Plasmids can replicate at a much higher copy number than the chromosome of the bacteria they inhabit which means any gene within a plasmid will be expressed to a greater degree than it would do if it was integrated into the bacterial chromosome.
Explain the process that allows foreign DNA to be inserted into a plasmid to form a plasmid vector
- Non-essential part of plasmid is linearised
- A multiple cloning site is inserted into the plasmid - It will only cut the plasmid at a specific part of its DNA sequence
- Foregin piece of DNA that you want to insert is amplified using PCR and then cut using same restriction enzymes found within the multiple cloning site so both the plasmid and DNA have compatible restriction sites
- Amplified foregin piece of DNA is then inserted into the plasmid where it will fit exactly at the point where the plasmid sequence has been cut as they have the same restriction sites
- Foreign DNA then ligated into plasmid via DNA ligase
- Non-essential part of plasmid is re-circularised
What is a multiple cloning site?
A piece of DNA containing a number of specific restriction enzyme sites
How can the production of a plasmid vector using recombinant DNA be used to prouce recombinant proteins?
- Plasmid vector is reintroduced into the bacteria where the plasmid came from, e.g. E.coli
- Recombinant bacteria is then placed onto an agar plate containing an antibiotic that the plasmid confers resistance to
- Vector contains a particular antibiotic resistance gene (selectable marker). This means that only the recombinant bacterial cells that contain the plasmid will be resistant to that particular antibiotic and be able to grow
- Recombinant bacteria is then grown and forms colonies which are then isolated
- The colonies are then used to confirm whether the foreign DNA has been inserted into the plasmid correctly by using restriction mapping
- Colonies are grown further and then cultured before the protein product that the bacteria of these colonies produce is purified
What are some of the uses of plasmid vectors?
- Allows for the expression of a recombinant gene in a living organism of choice
- Can be used to add or modify control elements of a gene - Can also make a gene inducible (ability to be activated or inactivated in response to a stimulus) or express it to high levels on demand
- Can alter the properties of the gene product - E.g Can make the expressed protein be secreted extracellularly
- Make the gene product produced from it useful as therapeutics
What are some examples of recombinant proteins used in therapeutics and what diseases are they used to treat?
- Human insulin - used to treat diabetes
- Interferons - α & β – used to treat viral Hepatitis or MS (multiple sclerosis)
- Erythropoietin – used to treat kidney disease, anaemia
- Factor XIII – used to treat haemophilia
What are some examples of recombinant antibodies (biologics) being used in therapeutics and what dieases are they used to treat?
- Synagis (Humanised anti-RSV IgG1 antibody) - Used to treat Respiratory Syncitial virus
- Herceptin (Humanised Anti-HER-2 IgG1 antibody) - Used to treat HER-2 positive breast cancer
What control elements are required to be in a plasmid vector for a desired gene within that vector to be expressed in a prokaryotic system?
- Coding region of the gene you want to be expressed
- Gene/s that code for the shine-dalgarno sequence
- A prokaryotic promoter
- A prokaryotic transcriptional terminator at the end of the gene coding sequence
How can the Lac operator and Lac repressor be used to produce an inducible promotor within a plasmid vector?
- Insert the Lac operator gene upstream of the promoter region of the foregin gene within the plasmid vector
- Introduce the Lac repressor gene within the plasmid vector
- Lac repressor protein produced from the expression of the Lac repressor gene will bind to the Lac operator preventing the transcription of the foreign gene.
How is a desired gene expressed under the control of a Lac operator/repressor inducible promoter within a plasmid vector?
- You add a lactose mimic (IPTG) to prokaryotic culture.
- The IPTG will bind to the Lac repressor protein causing it to change shape.
- This means it can no longer bind to the Lac operator thus allowing the RNA polymerase to transcribe the desired gene
What are the differences between the control elements required for a eukaryotic plasmid vector compared to a prokaryotic plasmid vector?
- In a eukaryotic plasmid vector instead of having genes that code for the shine-Dalgarno sequence there are genes that code for the Kozak sequence.
- Introns can also be present within a eukaryotic vector unlike a prokaryotic one
- Instead of having a prokaryotic terminator at the end of the sequence a eukaryotic vector will have a eukaryotic terminator
How can you improve the ability to purify a recombinant protein produced by a plasmid vector?
- To improve the ability to purify the protein produced you can add additional sequences to the 3’ end of the coding sequence just before the stop codon.
- This is called 3’ gene fusion
Describe a second way in which the ability to purify a recombinant protein produced by a plasmic vector can be improved
- Insert the sequence that codes for an entirely different protein into the coding sequence, e.g. Glutathione S transferase (GST), and turn your desired protein into a chimeric protein.
- Once the protein is produced you would use an antibody attached to an affinity column to separate the desired protein from the bacteria as the antibody will bind with the GST present within the desired protein
