Lecute 16 Flashcards
What colour will these be on a ampicillin, X-gal, ITPG
B N N W N
pUV vectors summary
- plasmid vector used for cloning
- ampicillin resistance
- blue/white selection
- need to use pUC vector with a compatible E. Coli strain e.g DH5-alpha
How can we adapt our plasmids vectors for the uses we want them for
What type of plasmid vector should we use?
Idk
what must a plasmid vector have
- origin of replication (must be capable of replicating indipendently in a cell)
- selectable marker
- multiple cloning site
Decisions in cloning a gene, what DNA do we use to express a protein
Use the gene we want to express
Decisions in cloning a gene, when wanting to express protein, what vector do we use?
Use expression vector
To express the cloned gene product we can use a…
Promoter
(Adapt basic plasmid structure and put a promoter in front of the multiple cloning site)
RNA polymerase will then bind the promoter to drive transcription and translation
What kind of promoter can we use to control the expression of expression vectors?
- using the Ptac promoter - induced by IPTG
(Can use IPTG to activate the Ptac promoter)
What do we use to produce large amounts of the cloned gene product?
We can use strong promoters
T7 promoter (comes from bacteriophage T7 which takes over the whole cell, take advantage of that)
What do we do when we want to purify out the protein expressed ( purify the gloved gene product)
- can add on a Tag and a cleavage site
- when its expressed it will make the cloned gene product but added onto it is a cleavage site and a tag
What is a tag? How does a tag work in purifying protein ?
A tag is something that we can use to bind to something else
- pass cell lysate (protein with tag attached) through a column
- the tag attached to our protein that we want to isolate will bind the column
- can then purify the protein from the column
- can then remove tag from protein using cleavage site (sometimes tag can interfere with how the protein works)
How can you purify a protein to be without GST tag?
Using PreScission protease
What if we want to visualise the protein we are expressing
- use GFP
- it is a green fluorescent protein (isolated form jelly fish, glows under U.V light)
Look at student example on the slides
What if we are interested in the promoter or enhancer region of a gene? UNDER WHAT CIRCUMSTANCE IS A GENE EXPRESSED? What DNA do we use?
Use the promoter of the gene
What if we are interested in the promoter or enhancer region of a gene? UNDER WHAT CIRCUMSTANCE IS A GENE EXPRESSED? What vector do we use?
Use expression vector with reporter gene
We can clone the promoter of “gene X (e.g lacZ GFP) in front of a reporter gene
- we can then test for different conditions for activation / repression of the promoter
What do we use to detect lac Z expression
X-gal
Not sure if need to know
Gateway cloning
- does not use restriction enzymes
- uses a site specific recombination system used by lambda phage
- insert DNA recombines and replaces the destination vector fragment
- ccdB gene fragment is contained within the destination vector. CcdB is toxic to cells so only those that have an insert replacing this gene can survive
- (need to recombine in the clone from the entry clone into the destination vector) we then select for expression clone using ampicillin and thus only expression clone will remain
- once an entry clone is made the ‘GENE Cassette’ can easily be moved into other vectors
Main features of gateway cloning
- based on lambda recombination system
- no restriction enzymes
- no ligation
- clone into an entry vector first
