exam 1 - genetics lab lec

lab 4 so far

lacl

makes the repressor ; separate from the operon

P - promoter

RNA Polymerase binds here to start transcription

O - operator

repressor binds here to block transcription

lacZ (makes what?)

makes B-galactosidase: breaks lactose into glucose + galactose

lacY (makes what?)

makes permease: transports lactose into cells

lac A (makes what)

makes transacetylase

Operon

structural genes controlled by one promoter

polycistronic mRNA

one mRNA translated into multiple proteins

negative control

controlled by a repressor

inducible

an inducer turns expression on

constitutive

expressed wtih/without inducer

affinity

binding strength

gene regulation

saves energy/resources by making proteins when needed

no inducer what happens

repressor binds operator → transcription blocked → operon off

inducer present

repressor inactivated→ cannot bind operator → RNA polymerase → transcribes genes → proteins made

-RNA polymerase binds P ; repressor binds O

I-

nonfunctional repressor - constitutive expression

O^c

Repressor cannot bind altered operator - constitutive expression

I^S

Super repressor cannot be inactivated by inducer - OFF with normal operator

P-

Rna polymerase cannot bind - no transcription

Z-

nonfunctional permease - no functional permease

combined mutation rules

• P- : blocks transcription even with I- or Oc

• Oc: prevents even a supperrepressor from binding

• Z-: acfects B gal; Y- affect permease (check them separately)

• transcription can occur even when the resulting protein is nonfunctional

IPTG

inducer - lactose analog that switches on a normal lac operon

SDS

detergent that opens/lyses cells, making enzyme accessible

ONPG

artificial substrate for B-gal ; cleavage produces a yellow product

Z Buffer

maintains suitable pH/reaction conditions

37 C

suitable temp for induction and B-Gal activity

water in -IPTG tubes

keeps volumes equal

bacteria

source of B gal

order

bacteria + buffer ± IPTG → incubate → SDS/mix → ONPG → incubate → observe color.

IPTG WHAT SDS WHAT ONPG WHAT

IPTG INDUCES

SDS OPENS

ONPG DETECTS

clear without IPTG, yellow with IPTG?

inducible : wild type or Y- , with other parts normal

Yellow with and without IPTG?

Constitutive: I- or Oc, with functional P and Z

clear with and without IPTG?

possible P-, Z-, or Is with normal operator

forgot bacteria or onpg

no yellow

forgot IPTG

wild type stays uninduced; constitutive mutants may still turn yellow

forgot SDS or adequate incubation?

weak/absent yellow