PART 3: Enzyme Assays

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PART 3 Enzyme Assays Recombinant E. coli
(lac operon minus host) carrying 2 plasmids
1. Lac repressor protein expression plasmid
(pWB100 series) 2. b-gal expression
plasmid with lac operator (pWB900
series) b-gal assay - how much b-gal is
expressed? REPRESSION If mutant
repressor binds to operator, then b-gal
not expressed NON-repression If
binding between repressor/operator is
weak, then will see b-gal expression What
does this tell you about the orientation of
the recognition helix?
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?-Galactosidase assay protocol Note Do not
forget to record the A600 of the chilled cells
prior to assay.   Mix the reaction components
together in the following order in 2mL microfuge
tubes 725 ?L Z buffer 75 ?L well-mixed cells (or
LB broth for blank reaction) 40 ?L chloroform 20
?L 0.1 SDS solution  
Vortex vigorously for 5 seconds. This is
important to permeabilize the cell
membranes!   At time zero Add 400 ?L
o-nitrophenyl-?-D-galactoside (ONPG) solution, 4
mg/mL in Z buffer. Mix gently.   Add the ONPG
solution at fixed time intervals of 15 or 20
seconds so that the timing of each reaction can
be precise for all tubes.   Allow the assay tubes
to incubate at room temperature until a pale
yellow color develops in some of the tubes.
About 5-15 minutes will probably be suitable.
Typically 10 minutes has been adequate. If
readings are too high, then re-do the assays and
incubate for 5 minutes. If readings are too low,
then re-do the assay with a 15 minute incubation
time.   Then, after the incubation time, stop the
reaction by adding 500?L 1M Na2CO3 and mixing
gently. This must be added at the appropriately
timed 15 or 20 second intervals. The increase in
pH caused by the addition of the sodium carbonate
will make the yellow color more
intense.   Centrifuge for 30 seconds to separate
the chloroform.   Using water as reference, read
the assay solutions at two wavelengths, 420nm and
550nm. Be sure to avoid the chloroform bubble in
the bottom of the tube when placing the reaction
solution in the spectrophotometer cell. Note
When making calculations, be sure to subtract the
two blanks, the assay blank and the turbidity
blank. The turbidity blank is obtained by
multiplying 1.75 times the OD at 550nm. The
assay blank obtained by using LB instead of cells
in the assay protocol and read the absorbance at
420nm. The corrected absorbance at 420 nm the
uncorrected absorbance at 420nm minus the assay
blank and the turbidity blank.
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YELLOW indicates that Bgal is expressed (NO
Repression) CLEAR indicates Bgal is NOT expressed
(REPRESSION)
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(Wt variation)
(Wt variation)
Wildtype Lac Repressor
Wildtype Operator Sequence
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X
(Wt variation)
(Wt variation)
REPRESSION Repressor Binds Operator No Beta Gal
expression
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Repression
 
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(Any variation)
(Delta variation)
Delta Lac Repressor Completely lacks
recognition sequence
Any Operator Sequence

• 100 B-gal Expression for that operator plasmid
• LacR missing recognition helix
• will not bind any Operator
• Control to let you know how much Bgal is produced
  by the operator plasmid

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Repression
 
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(Wt variation)
(Mutated variation)
EXPRESSION Repressor does NOT bind
Operator Beta Galactosidase Expressed
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Repression
Exp
 
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X
(Mutated variation)
(Mutated variation)
SUPPRESSOR PAIR REPRESSION Mutated Repressor
Binds Mutated Operator
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0
Exp
0
Exp
 
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Rep
Exp
Rep
Exp