L16 - Non-Genetic Analysis of Gene Function Flashcards
What sort of promoters do expression vectors use
Bacteriophage
Why are bacteriophage promoters used in expression vectors
To drive high levels of expression (high levels of RNA synthesis)
Why must the promoters of expression vectors be inducible
Else the bacteria would die from synthesising too much of protein
What is the type of promoter present in expression vectors
Inducible
In what two ways can the promoter be induced in an expression vect
Heat shock or chemical treatment
What occurs to the bacteria after they have been induced to make alot of protein specific antibody
Harvested in a centrifuge and cells are lysed to make a crude extract
What do expression plasmids often include
An epitope tagging system that allows for rapid and efficient purificaiton of the portien
How is the epitope added to the protein
Fused in frame
What are epitope tags
Peptides for which antibodies are already available
Describe the steps in antibody affinity chromatography
Antibodies covalently bound to beads in the column
Pour through extract - target proteins will bind to the antibodies
Elute using a pH 3 buffer and resulting fraction will be the target protein
Why must a pH 3 buffer be used in antibody affinity chromatography
To disrupt the interactions between the target proteins and the antibodies
Describe the steps in making a protein specific antibody
Protein (gene) –> Vector –> Bacteria
Grow the bacteria which will synthesise the protein
Purify the protein
Inject the protein into a rabbit several times over a 6 month period
Purify the antibody from the serum of the rabbit
Why is the protein injected into the rabbity
Rabbits own immune system will produced antibodies against the protein - these can be harvested
Antobodies can be tagged with tags and dyes in a process known as
Conjugation
What is the main method of detection used
Flourescence is the main method used
What can antibodies be used to detect
Subcellular localisation of the proteins and which tissues are expressing those proteins
What are two commonly used conjugates - state the colour change that would be observed
Alkaline phosphatase
- Substrate would turn blue
Horseraddish peroxidase
- Substrate would turn brown
Why would two antibodies be used
Many secondary antibodies (from a company) would bind to the primary antibody this would amplify the signal
Describe the process for using antibodies
How can this process be varied for a different view/
Chemically fix (using formaldehyde) the organism / tissue to stabilise the structure Incubate with the conjugated AB AB will bind to the target protein Wash off the eexcess antibody Whole mount produced - see signal
Can take a slice of the tissue/organism in order to obtain a view from a different persective
SECTIOn
Describe the steps required for a mRNA in-situ analysis
Purify vector containing cDNA of interest
Synthesis the antisense strand and incorporate epitope tagged nucleotides
Incubate in the embryo with the probe
Anti sense probe will hybridise with the endogenous mRNA
Wash
RNA detection systems usually use a blue substrate - alkaline phosphatase
Describe the expression of bicoid mRNA
What technique would be used to visualise this expression
Expression is high anterior, no expression anywhere except for the extreme anterior
Describe the expression of bicoid protein
Expressed in a decreasing gradient from anteior to posterior - highest anteiorly and lowest posteriorly
What wavelength is used to excite GFP
475 nm
What wavelngth of light is emitted by GFP
510 nm
Why is emission wavelength longer than exictation
Because less energy in emission - electron loses energy when it changes its energy state
What are the three steps of genergating a GFP transgenic line
Clone the entire gene with all of the regulatory elements into a plasmid
Genetically engineer GFP onto the end of the last exon (gene fusion) or replace the gene (reporter)
Integrate the GFP fusion gene back into the organisms genome
The integrate gene is known as a
Transgene
Describe how a gene fusion is achieved
Bolt the GFP gene onto the end of the last exon
Describe how a reporter construct is formed
replace the gene with the GFP gene
What is a reporter construct used for
To visualise expression of a gene (mRNA)
How is GFP integrated back into the organism
Involves microinjecting into the one cell zygote, DNA then randomly integrates into the genome
What are three uses for transgenic GFP lines
1) Follow gene expression in the whole animal
2) Follow subcellular location of a protein
3) To follow behaviour of the cells - mark specific cells so that they can be distinguished from their neighbouts
What situation would a GFP reporter constrcut be utilised for
To follow the behaviour of cells in vivo - marking cells
What situation would a GFP fusion protein be used for
To follow the subcellular location of the protein
