Lecture 19 Flashcards
1
Q
What are the functions of IPTG in the pUC vector?
A
It must be added to induce the LacZ gene
- It binds to the lac repressor
- > acts like allolactose
- > not broken down by Beta-gal
2
Q
Describe an expression vector
A
- RNA pol will bind the promotor to drive transcription and translation
- Can control expression using the Ptac promoter - induced by IPTG
- To produce large amounts of clone we can use a strong promoter (e.g. T7)
3
Q
Describe purifying the cloned gene product
A
- Pass cell lystae through a column
- > the tag attached to the protein of interest will bind to the column - Purify protein from the column
- Remove TAG from protein
- >incase it interferes with the protein
4
Q
Describe TAG binding
A
It will bind very strongly to something else
5
Q
Does Green Fluorescent protein interfere with the gene product?
A
No
6
Q
How can we find out what circumstances a gene is expressed?
A
Can clone a promotor of ‘Gene X’ in front of a reporter gene
->test different conditions for activation/repression of X
7
Q
Describe Gateway Cloning
A
- First clone into an entry clone (this makes it easier to move between different vectors)
- Insert DNA recombines and replaces destination vector fragment
8
Q
What does Gateway Cloning not use?
A
- Restriction enzymes
- No ligation
9
Q
What does the destination vector fragment have?
A
It has ccdB which is toxic so only cells that have the insert replacing this gene will survive
