Recombinant DNA Technology and Cloning Vectors Flashcards
What is the key to studying genes?
The experimental manipulation of extra-chromosomal DNA (DNA found off the chromosomes, either in or out the nucleus of a cell) and its use in recombinant DNA technology (DNA formed from multiple sources, creating sequences that wouldn’t be in the genome) is key to our ability to study genes.
There are various types of extra-chromosomal DNA tools that we refer to as vectors (used to transfer DNA into a biological system).
List the 4 examples of recombinant vectors.
PLASMIDS:
- Found in many but not all bacteria.
- Generally Have a restricted host range.
- Are transferable by various means including transformation (mainly done experimentally like this) and conjugation.
PHAGES:
- Lambda – bacterial viruses
- Transfer of antimicrobial resistance through a mechanism called transduction
- Restricted host range, their restriction and ability to lyse bacteria means they have been used as antibacterial agents.
VIRUSES:
- Non-primate Lentiviruses –vectors used to integrate DNA in mammalian cells
- Baculoviruses –vectors used in combination with recombinant expression in insect cells (a eukaryotic expression system).
ARTIFICIAL CHROMOSOME:
- Yeast Artificial Chromosomes (YACs) – introducing large segments DNA (including promoters and introns).
- YACs are similar conceptually to plasmids but are much bigger and are restricted to yeast
plasmids.
What are plasmids?
They are discrete circular double stranded DNA (dsDNA) molecules found in many, but not all bacteria used as tools for introducing DNA into both eukaryotes and prokaryotes. They are a means by which genetic information is maintained in bacteria and passed vertically do their progeny.
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), facilitated by conjugation. However conjugative plasmids are NOT normally used and recombinant vectors are therefore, not exchanged horizontally. This is an important feature.
What are vectors and what are some of their uses?
Vectors are a cut down version of naturally occurring plasmids & are used as molecular tool to manipulate genes.
- Cloning a gene (or disease causing variant gene) to produce a recombinant protein in a biological system (E.g. a bacterium in large quantities.)
- Used to mutate a gene and understand the functions of parts of a protein or the effects of a specific mutation on protein structure/function.
- Used to insert promoters in front of reporter genes –> to better understand the regulatory mechanisms of a gene promoter.
- Used in 2 component systems to understand the interaction and association of different gene products in a biological system (E.g. - the yeast 2 hybrid system).
What are the characteristics of a plasmid (vectors)?
- 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 or be maintained.
- Are often modified to replicate at high multiplicity (copy number) within a host cell, allowing genes to be expressed to much higher levels and the recovery of greater amounts of DNA and/or gene products for experimental use
- Contain selectable markers (E.g. - antibiotic resistant genes like ampicillin)
- Most 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:
o Make it secreted extra-cellularly or into. the periplasmic space
o Fuse it to a peptide tag or other protein.
o 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.
- Many of these drugs are humanised antibodies.
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 and range of restriction sites).
What control elements are required for expression in bacteria?
- Gene coding sequence is insufficient –> in bacterium we don’t want UTRs, intronic or regulatory sequences such as promoters or enhancers (i.e. introns can be processed in prokaryotes and eukaryotic regulatory sequences won’t work)
- Shine-Dalgarno sequence –> RBS (ribosomal binding site) found around 8 nucleotides before the start codon in the RNA in prokaryotes (AUG)
- Strong Bacterial promoter to initiate transcription and must be added at the 5’prime end of the transcription unit.
- Transcriptional terminator –> to allow polymerase to end transcription and release message.
All ‘parts’ are needed for successful expression.
The recombinant gene can be expressed in using a Constitutive or Inductible Promoter. Describe them.
CONSTITUTIVE - always switched 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 - use molecular switch to switch a promoter on and off:
(a classic one used in prokaryotes is adapted from the promoter of the lac operon)
- Allows large cultures to be grown without expressing the foreign protein.
- Induced in response to a defined signal —> The production of a toxic protein has little affect on the growth of the culture –> allowing the protein to be produced.
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.
This system can regulate any gene by placing a lac operator (lacO) upstream of its transcriptional start.
In an artificial system, the lac Operator is de-repressed experimentally by addition of a lactose mimic called IPTG.
What are the requirements of the DNA insert?
- The DNA must be easy to manipulate – to cut and re-join to with other DNA, add restriction sites using PCR or other methods
- Copy of the coding sequence – generated by e.g. PCR
- Must contain the start and 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
What are some of the features a Bacterial Plasmid must have in order to be used as a
vector?
The selection of the vector is important - it must have the correct features for inserting the gene selecting for recombinants.
- It may require promotors or other elements in the correct place for e.g. - the bacterial promoter, multiple cloning sites (in which the gene will be inserted), a variety of restriction sites (Xba I etc) where we may cut and linearize it.
- It should also have a bacterial transcriptional terminator.
- When the sequence is transcribed, it can only be translated into a protein, if the sequence contains the correct in-frame start and stop codons upstream.
- There should also be a Shine Dalgarno sequence (a ribosomal binding site in bacterial mRNA, generally located around 8 bases upstream of the start codon AUG). The RNA sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ribosome with the start codon.
How can Bacterial Plasmids be used as vectors?
Memorise this
We can use a plasmid vector & insert a recombinant gene into it.
- The vector and PCR amplicon (from coding sequence of gene) must be cut via restriction enzymes to produce compatible ends.
- These are then joined together by ligation using a DNA ligase.
- The recombinant plasmid made can then be artificially transduce bacteria, where the plasmids will replicate and be maintained in the presence of a selectable marker such as ampicillin.
- We can then pick individual colonies (clones) and grow these up in bulk to produce recombinant proteins in our bacteria (where we can choose to for e.g. purify the protein produced, investigate its properties/function or alternatively develop and produce therapeutics).
- The protein is then induced by addition of a lactose mimic IPTG to produce recombinant protein.
