Genetics News

1
Genetics News

• Seminar Monday, Oct 30John RyanImmunology,
  asthma
• Printing problems

2
Topics for today
Phenotypes of strains diploid for lac
Model for positive regulation of lac operon
Experimental test of model
3
Predict the Lac phenotype
ß-galactosidase phenotype I inducible by
IPTG C constitutive activity N no
activity
Growth Lac can grow on lactose Lac-
cannot grow on lactose
Chromosome Plasmid
Phenotype
I P O Z Y I P O Z Y
4
Predict the Lac phenotype
ß-galactosidase phenotype N no activity (or
very low)
- lactose
X
- lacZ is dead
lactose
X
X
5
Predict the Lac phenotype
ß-galactosidase phenotype N no activity (or
very low)
- lactose
- lacZ is dead
X
- promoter is dead
lactose
X
X
X
X
X
6
Predict the Lac phenotype

ß-galactosidase phenotype N no activity

- lactose
- lacZ is dead- promoter is dead

lactose
I inducible by IPTG
- good lacZ, promoter
- good lacI

- good operator
7
Predict the Lac phenotype
ß-galactosidase phenotype N no activity
X
- lacZ is dead- promoter is dead
X

I inducible by IPTG
- good lacZ, promoter- good lacI- good operator

C constitutive activity
- good lacZ, promoter
- bad lacI
- OR bad operator
8
Predict the Lac phenotype
Growth Lac can grow on lactose
- good lacZ
- AND good lacY
lacZ
lacY
9
Predict the Lac phenotype
Growth Lac can grow on lactose
- good lacZ
- AND good lacY
X
X
Lac- cannot grow on lactose
- bad lacZ
lacZ-
10
Predict the Lac phenotype
Growth Lac can grow on lactose
- good lacZ
- AND good lacY
X
X
X
Lac- cannot grow on lactose
- bad lacZ
- OR bad lacY
lacY-
11
Lac Phenotype Strategy
Check trans-acting regulatory factors

• Repressor
• CAP
• RNA polymerase

Check cis-acting regulatory factors
Whats a trans-acting factor?Whats a
cis-acting factor?
SQ8. Does a start codon mutation act in
cis or trans?
SQ9. Would a promoter mutation act in
cis or trans?

• Promoter
• Operator

12
Lac Phenotype Strategy
I- P O Z Y F( I P Oc Z- Y)
Check trans-acting regulatory factors
-

• Repressor
• CAP
• RNA polymerase

X
X
Check cis-acting regulatory factors
X
- -

• Promoter
• Operator

ß-Gal phenotype?
Growth?
13
Lac Phenotype
I- P O Z Y F( I P Oc Z- Y)
ß-Gal phenotype?
-
lacZ repressed
(lacY constitutive)
- -
Without lactose
14
Lac Phenotype
I- P O Z Y F( I P Oc Z- Y)
ß-Gal phenotype?
-
lacZ induced
(lacY constitutive)
INDUCIBLE
Growth phenotype?
ß-gal made
- -
Permease made
GROWTH
With lactose
15
Positive Regulation of lac operon
State

• Plenty of glucose

Response

• Eat it

• Avoid other sugars

Symbol of state

• Low cAMP

glucose
lactose
other sugars
16
Positive Regulation of lac operon
State

• No glucose

Response

• Eat other sugars

Symbol of state

• High cAMP

lactose
other sugars
17
Positive Regulation
X
X
crp-
Test of modelWhat happens if...
X
X
Low expression of lac operon
18
Positive Regulation
X
Test of modelWhat happens if...
X
X
Low expression of lac operon
(Why defects lead to low expression?)
19
Positive Regulation
X
Test of modelWhat happens if...
X
X
X
Low expression of lac operon
20
Positive Regulation
Protein Regulation Target Acts on
Influenced by
Lac repressor CRP
Negative operator lac operon
lactose
Positive
CRPbinding site
many operons
cAMP
21
Positive RegulationSQ14 Superimpose cAMP
ß-gal on growth curve
When is glucose present?
- glucose
ß-gal
When is cAMP present?
cAMP
When is ß-gal present?
glucose
22
Test of Positive Regulation - SQ15
Absence of inducer, ß-gal activity?
Presence of IPTG, ß-gal activity?
Presence of IPTG and glucose, ß-gal activity?
Grown in lactose glucose, diauxic effect?
23
Test of Positive Regulation
SQ16Is diauxic growth apparent?
SQ17Is appearance of ß-gal repressed by glucose?
From Kimata et al (1997)
24
SQ22 How to make biosensor of heavy metals
Red on MacConkey plates