L16 - Non-Genetic Analysis of Gene Function

Question 1

What sort of promoters do expression vectors use

Answer 1

Bacteriophage

Question 2

Why are bacteriophage promoters used in expression vectors

Answer 2

To drive high levels of expression (high levels of RNA synthesis)

Question 3

Why must the promoters of expression vectors be inducible

Answer 3

Else the bacteria would die from synthesising too much of protein

Question 4

What is the type of promoter present in expression vectors

Answer 4

Inducible

Question 5

In what two ways can the promoter be induced in an expression vect

Answer 5

Heat shock or chemical treatment

Question 6

What occurs to the bacteria after they have been induced to make alot of protein specific antibody

Answer 6

Harvested in a centrifuge and cells are lysed to make a crude extract

Question 7

What do expression plasmids often include

Answer 7

An epitope tagging system that allows for rapid and efficient purificaiton of the portien

Question 8

How is the epitope added to the protein

Answer 8

Fused in frame

Question 9

What are epitope tags

Answer 9

Peptides for which antibodies are already available

Question 10

Describe the steps in antibody affinity chromatography

Answer 10

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

Question 11

Why must a pH 3 buffer be used in antibody affinity chromatography

Answer 11

To disrupt the interactions between the target proteins and the antibodies

Question 12

Describe the steps in making a protein specific antibody

Answer 12

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

Question 13

Why is the protein injected into the rabbity

Answer 13

Rabbits own immune system will produced antibodies against the protein - these can be harvested

Question 14

Antobodies can be tagged with tags and dyes in a process known as

Answer 14

Conjugation

Question 15

What is the main method of detection used

Answer 15

Flourescence is the main method used

Question 16

What can antibodies be used to detect

Answer 16

Subcellular localisation of the proteins and which tissues are expressing those proteins

Question 17

What are two commonly used conjugates - state the colour change that would be observed

Answer 17

Alkaline phosphatase
- Substrate would turn blue

Horseraddish peroxidase
- Substrate would turn brown

Question 18

Why would two antibodies be used

Answer 18

Many secondary antibodies (from a company) would bind to the primary antibody this would amplify the signal

Question 19

Describe the process for using antibodies

How can this process be varied for a different view/

Answer 19

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

Question 20

Describe the steps required for a mRNA in-situ analysis

Answer 20

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

Question 21

Describe the expression of bicoid mRNA

What technique would be used to visualise this expression

Answer 21

Expression is high anterior, no expression anywhere except for the extreme anterior

Question 22

Describe the expression of bicoid protein

Answer 22

Expressed in a decreasing gradient from anteior to posterior - highest anteiorly and lowest posteriorly

Question 23

What wavelength is used to excite GFP

Answer 23

475 nm

Question 24

What wavelngth of light is emitted by GFP

Answer 24

510 nm

Question 25

Why is emission wavelength longer than exictation

Answer 25

Because less energy in emission - electron loses energy when it changes its energy state

Question 26

What are the three steps of genergating a GFP transgenic line

Answer 26

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

Question 27

The integrate gene is known as a

Answer 27

Transgene

Question 28

Describe how a gene fusion is achieved

Answer 28

Bolt the GFP gene onto the end of the last exon

Question 29

Describe how a reporter construct is formed

Answer 29

replace the gene with the GFP gene

Question 30

What is a reporter construct used for

Answer 30

To visualise expression of a gene (mRNA)

Question 31

How is GFP integrated back into the organism

Answer 31

Involves microinjecting into the one cell zygote, DNA then randomly integrates into the genome

Question 32

What are three uses for transgenic GFP lines

Answer 32

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

Question 33

What situation would a GFP reporter constrcut be utilised for

Answer 33

To follow the behaviour of cells in vivo - marking cells

Question 34

What situation would a GFP fusion protein be used for

Answer 34

To follow the subcellular location of the protein