Term 2 Lecture 5: Expression Vectors And Gene Isolation Flashcards
The first step
Is often cloning a DNA fragment (usually a gene) into a bacterial plasmid.
DNA is verified correct by DNA fragment PCR and RE digestion+ electrophoresis testing and sometimes DNA sequencing
Next step
Move gene to another organism - usually a eukaryotic cell system (plant/animal/yeast)
Discover where in the organism that the gene is expressed (e.g. by making a transgenic organism)
Control gene expression e.g. only in particular cell type/when experimentally induced →to do this subclone the fragment into an expression vector
You may also want to express the protein and purify it.
Typical expression vector
Example: bacterial antibiotic resistance gene - allows expression vector to be grown in bacteria prior to introducing it to a plant or animal. The bacterial promotor drives expression
Features
Promotor: e.g. mammalian or plant promotor, could be constitutive/ tissue specific/ MCS (multiple RE sites for subcloning of gene into expression vector)
Antibiotic resistance gene: allows plant/ animal cells containing expression vector to be selected for (promotor drives expression of this gene)
Terminator: transcription stop sequence
Other examples of expression vector
E.g. mammalian reporter gene expression vector has an additional GFP (green fluorescent protein) gene
E.g. plant expression vector
Promotor such as CaMV
(cauliflower mosaic virus)
MCS
T-DNA
Antibiotic resistance
About 12kb in size
Terminator
More on expression vectors
Expression vectors are specific to host cells e.g. plant and animal vectors are different because:
-promotors are organism/cell specific i.e. can only be turned on where they are recognised by proteins required to initiate transcription (transcription factors)
- some sort of selection process is required to ID recombinant cells e.g. antibiotics work better on particular cells
- expression vectors often include reporter genes
How to isolate a gene : when gene sequence is known
PCR could be used to design primers from the gene sequence
Many genomes are now sequenced and annotated
This could be done in a 3rd year project over 5 weeks:
-genomic DNA (template) containing gene to be cloned combined with gene specific primers
-add dNTPs and PCR buffer and set up PCR reaction
- check gene isolation by gel electrophoresis
How to isolate a gene: When genome sequence is not known
Even if the genome you are working with has not been sequenced yet there may be a homologous gene or gene performing the same function in another species
Regions of homology can be used to design PCR primers
Bio informatic analysis, compare with another species by carrying out ‘alignment’ with homologous regions
Take a gene to use as a probe tool to isolate the gene from another species.
How to isolate a gene: when the genome sequence is not known but protein sequence is known
Align proteins by BLAST search - aligns aminos to find homology - so you can write out possible codons.
PCR primers can be designed by referring to genetic code e.g. a ll protein sequence has 256 possible primers (chosen by algorithm)
This online tool required you to enter the protein sequence and it provides all possible combinations.
The company synthesises a pool of primers which is received as a mix in a tube which the scientists add to genomic (target) DNA. Due to the specificity of PCT only one of the 256 primers will bind (the presence of the others doesn’t matter
How to isolate a gene: when the gene or protein is very short
Proteins of 500-5000 BP are considered short, they can be chemically synthesized.
E.g. signalling peptides are very short so rather than cloning them they get synthesised
IDT: upto 500bp costs £125 and takes 8 business days.
If PCR is unsuccessful
PCR may just be the start point because:
- may not be possible to amplify entire gene
-errors may be introduced by PCR - in this case PCR fragments can be used as a probe to screen a DNA library - Taq polymerase can be used to correct errors but this method is expensive and not 100% effective. - previously isolated genes from other organisms also work as probes
- errors in a PCR cannot be corrected
DNA library
Archive of all genetic material of an organism, cloned into individual plasmids.
A ‘library’ of plasmids in bacterial clones.
Genomic DNA cut randomly with restriction enzymes (typically with RE that give sticky ends) + plasmid cut by the same enzyme
Each plasmid contains a different fragment all transformed into bacteria to make a ‘library’
Can be plated out to form individual colonies
→ before creating a DNA library carry out a Southern blot to determine if the gene you are interested in can be detected by the probes you have
Southern blot
Layers:
Paper towel
Nitrocellulose paper
Gel
Sponge
Alkali solution and salt
The method was named after Ed Southern. Isolating total genomic DNA, digesting it with RE to fragment and then run on a gel.
Gel is blotted, top gel with a membrane of nylon or nitrocellulose to bind DNA irreversibly. Paper towels on top draw buffer up from the sponge through the gel carrying DNA fragments up onto the nitrocellulose paper.
Peel off membrane check all DNA has transferred by restraining the gel with ethidium bromide (should result in no stain confirming all DNA transferred)
DNA on Southern blot is immobilised and denatured ssDNA (due to alkali treatment.) Labelled ssDNA fragments (hyperladder) can be used to probe blot for complementary DNA fragments
However these fragments cannot be extracted they are permanently bound to the nitrocellulose.
Labelling DNA to make a probe
- you need a fragment of DNA to label usually a PCR fragment (short region of gene)
- heat to denature
- add primers - different to PCR, random 6 base sequences (hexamers) can be bought and synthesised to label random sections of DNA
- hexamers bind to sequences along the probe and act as primers if DNA polymerase is added to synthesise a second strand.
- chemical or radiolabel markers are used to visualise e.g. radiolabel (32P) dCTP
Southern blot → probe
Hybridisation reaction
Radiolabelled probe is hydridised to separate DNA
Seal nitrocellulose membrane (from Southern blot) in buffer with labelled probe and leave overnight to rock gently
Binding/hybridisation reaction: ss probe binds to complementary sequence on membrane
Next day hope to see probe hybridised to a specific band or fragment, detected depending on type of label e.g. for a radiolabel the fluorescent marker is detected by autoradiography - membrane placed in a n x-ray cassette covered by x-ray film which detects where radioactivity is
- should show a clear band or two which tells you that your probe can detect a fragment of DNA with your genomic DNA
Also:
RNA can be blotted and probed in the same way and this is called a Northern blot.
Proteins can be blotted and probed with an antibody and this is called a western blot
So seeing a band from a Southern blot confirms a DNA fragment or gene of interest is present in a sample of genomic DNA - to isolate the gene a DNA library needs to be made
→ you can’t isolate the gene from the blot as it is immobilised (cannot be removed) this process just confirms that it’s worth going to a DNA library to try to isolate the fragment
DNA libraries: 3 types
3 types: genomic DNA/ cDNA/ expression
In these lectures we cover plasmid libraries but DNA libraries can also be made in phage lambda to clone larger fragments
Process:
DNA is isolated
Digested by RE
Cloned into plasmids
Plasmids are then put into bacteria
