Lecture 19

Question 1

What are the functions of IPTG in the pUC vector?

Answer 1

It must be added to induce the LacZ gene

• It binds to the lac repressor
• > acts like allolactose
• > not broken down by Beta-gal

Question 2

Describe an expression vector

Answer 2

• 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)

Question 3

Describe purifying the cloned gene product

Answer 3

1. Pass cell lystae through a column
   - > the tag attached to the protein of interest will bind to the column
2. Purify protein from the column
3. Remove TAG from protein
   - >incase it interferes with the protein

Question 4

Describe TAG binding

Answer 4

It will bind very strongly to something else

Question 5

Does Green Fluorescent protein interfere with the gene product?

Answer 5

No

Question 6

How can we find out what circumstances a gene is expressed?

Answer 6

Can clone a promotor of ‘Gene X’ in front of a reporter gene
->test different conditions for activation/repression of X

Question 7

Describe Gateway Cloning

Answer 7

• First clone into an entry clone (this makes it easier to move between different vectors)
• Insert DNA recombines and replaces destination vector fragment

Question 8

What does Gateway Cloning not use?

Answer 8

• Restriction enzymes

- No ligation

Question 9

What does the destination vector fragment have?

Answer 9

It has ccdB which is toxic so only cells that have the insert replacing this gene will survive