GENE EXPRESSION

Gene Expression
Controlled at many levels.

Transcription

mRNA Processing

Post - translational

Transcription control
Basic concept provided by Jacob and Monod
Distinguished between cis-acting sequences and
trans-acting products.
c.a.s are sites on DNA
Are not transcribed and only function as DNA sequences

t.a.p are diffusible

Protein products of genes that function elsewhere from the site
of their production.
Structural genes
- Regulatory genes

OPERON

proteins with related functions

Catabolite Repression (aka glucose repression)

II
I

Bacteria growing on two different sugar sources

display a biphasic growth pattern

Lac Operon

Cis-acting sequence - Promoter, Operator and Terminator

Trans-acting products Structural genes
-lac Z -galactosidase
-lac Y lactose permease
-lac A Transacetylase
- Regulatory gene
-lac I Repressor

Organization of the promoter-operator region of the lac operon.

CAP Catabolite Activator Protein

Gene Expression
Induction Synthesis of enzymes in response to
a specific inducer.
Repression Inhibition of transcription and
synthesis of certain enzymes when their
products are present.
Performed by inducers and co-repressors.
Gratuitous inducers
IPTG

Negative regulation of the lac operon

Substrate Induction

Positive regulation of the lac operon

cyclic AMP catabolite activator protein (CAP/CRP) turns up the volume

Catabolite Repression

Lac operon is negatively regulated by the lac repressor

and positively regulated by the cAMP-CRP complex
High glucose
No lactose

Low glucose
No lactose

Low glucose
High lactose

High glucose
High lactose
-Basal level exp

Lac Z Functions

Lactose: Galactose (b 14) Glucose

Allolactose: Galactose (b16) Glucose

Lactose is converted to allolactose by b-galactosidase by
alternative rxn to the hydrolytic one

Gratuitous Inducer
IPTG - Allolactose analog used for induction of lac operon

Nonmetabolizable
Transported easily

Metabolic Analog
ONPG - Lactose analog used to assay -galactosidase activity
Used in combination with inducer (i.e. IPTG)
Colorimetric assay
ONP Spectrophotometric substrate 420nm

ONPG (orthonitrophenyl -D-galactoside)

Metabolic Analog
X-gal Used to detect the presence of LacZ gene product

Basis for the blue/white screening of colonies

Used in combination with the inducer (IPTG)
Transformation control or Reporter gene

X-gal bromo-chloro-indolyl-galactopyranoside (BCIG)

Reporter gene

Gene expression assay

Promoter Assay

Today
We will observe lac operon induction and
catabolite repression
Inducer IPTG
Enzyme substrate ONPG
Repressor Glucose

15 ml of log phase
E. coli to each flask

Transfer 1ml
to tube U
before the
addition of
IPTG

UG

IPTG

IPTG
+
Glucose
Transfer 1ml
to tube UG
before adding
IPTG and glucose

Add chloroform & SDS, Vortex each tube for 5 seconds and place on ice.

IPTG

IPTG
+
Glucose

IPTG

150l

150l

Glucose

500l

-Return to 30 oC incubator.
-Transfer 1ml samples to labeled test tubes at designated times.
-Collect induction samples at 2, 4, 8, 16, 24, 32 and 48 min intervals.
-Collect repression samples at 10 and 45 minute intervals only.
-After every collection Vortex tubes 5 seconds and place on ice.

Labeling glassware
4

16

U/UG = time 0

24

+45G

+10G

32

48

-gal Assay
-Turn on the spectrophotometer.
-Label 11 tubes as before plus one tube for blank.
-Add 1.5ml ONPG assay mix to each tube (all 12).
-At time 0, add 0.15ml of cell extract to their corresponding
tubes.
-To the blank tube, add 0.15ml of LB.

-Mix the tube contents and place in 30oC bath.

-Stop the reactions together when the reaction has turned
yellow by adding 1.5ml of Na2CO3 to each tube including the
blank.
-vortex to mix and read absorbance at both 420nm and 550nm

Absorbance Correction
z Abs420 O-nitrophenol + cell debri
z Abs550 Cell debri only
z Light scattering at Abs420 is proportional to that
at Abs550 by
y O.D. 420 light scattering = 1.75 X Abs550

z Therefore corrected absorbance by onitrophenol at 420nm is

y Abs420 (1.75 X Abs550)