Workshop 4

Question 1

What is an operon ?

Answer 1

Operon: All genes that are transcribed into the same mRNA plus any adjacent cis-acting sites involved in transcription or regulation.

Question 2

What is a regulon ?

Answer 2

Regulon: When more than one operon is under the control of a single regulatory protein, these operon are called regulon (maltose utlization).

Question 3

What are lacZ, lacY and lacA ?

How are they organized on the bacterial chromosome ?

Answer 3

3 structural genes are clustered together on 
chromosome
lacZ: ß-galactosidase
lacY: lactose permease
lacA: galactoside transacetlyase

Question 4

What is lacP ?

Answer 4

lacP: promoter needed to transcribe the region as as single polycistronic mRNA

Question 5

What is lacO ?

Answer 5

lacO: operator site involved in transcriptional regulation

Question 6

What is lacI ?

Answer 6

lacI: repressor protein

Question 7

What is CRP (or CAP) ?

Answer 7

CRP protein (also CAP protein): catabolic gene activation protein.

Question 8

What is an inducer ?

Answer 8

A susbtance that induces enzyme synthesis.

Question 9

How many binding sites does the repressor protein have ?

Waat do these bind to ?

Answer 9

The repressor protein has two binding sites

• one for the operator
• one for inducer

Question 10

What is the structure and function of the repressor protein ?

Answer 10

• Functional repressor is tetramer
• Binding to o2 and o3 or o1 and o3
• Promoter and CAP binding region
• lac promoter typical sigma70 promoter (-10 & -35 region)
• Bends DNA –> helps to prevent RNA polymerase binding

Question 11

How is the lac operon regulated when lactose is absent ? - present ?
What about when glucose is present ?

Answer 11

Lactose absent :
- No lactose (inducer)
- repressor protein is continously synthesized
- active repressor sits on operator site and blocks way of RNA polymerase
Lactose present :
- small amount of lactose converted in allolactose
- repressor inactive
- RNA polymerase can bind to promoter region
3. Glucose present
- cAMP level is low
- CRP protein does not bind
- RNA polymerase keeps falling off promoter site

Question 12

What we performed alkaline lysis, what was the composition of our solutions I, II and III ?

Answer 12

Solution I :
50mM Tris-HCl, 10mM EDTA, 100 μg / ml RNase A pH 8.0
Solution II : 
200 mM  NaOH
1 % SDS
Solution III :
2.8M KAc pH 5.1

Question 13

What is the threshold (in cfu/ug) for competent bacteria (using transformation) ?
Is it more efficient to introduce DNA chemically ? - electrically ?

Answer 13

~ 10^6 cfu/ug
Chemically : 1-3e9 cfu/ug
Electrocompetent : 2e10 cfu/ug

Question 14

What is the function of EDTA ?

Answer 14

Metal ions scavenging.

Question 15

What is the function of SDS ?

Answer 15

Protein denaturation.

Question 16

What is the function of KAc ?

Answer 16

Potassium acetate is used to precipitate dodecyl sulfate (DS) and DS-bound proteins, allowing the removal of proteins from DNA. It is also used as a salt for the ethanol precipitation of DNA.