Jesse Ruben BMB 442

P: Roman Verner Section 1

Lac Operon
Introduction
Understanding the mechanisms that regulates protein synthesis in a cell is very
important and in this lab we experiment with the manipulation of genes expression of cells
during different levels of glucose and lactose environments. The CAP binding site, promoter,
and operon regulates specific transcriptional control, mRNA synthesis, under different
environmental conditions, which would allow repression or induction binding of the RNA
polymerase due to promoter and repressor complexes. The Lac genes would encode the mRNA
for B-galactosidase (B-Gal), which breaks ester bonds between lactose and produce glucose and
galactose for the cell to use. The second mRNA in which the Lac gene expresses is lactose
permease, which allows the lactose to enter the cell and metabolize into the two monomer
sugars and allolactose, which bind to the Lac repressor and further inactivate its binding to the
operon, thus allowing RNA polymerase to bind. These enzymes are normally repressed in the
cell causing the gene to be repressed.
As we see in the Lac metabolic pathway during high levels of glucose, the cell is in a
catabolite repression state, where presents of the cyclic AMP are low and its binding to the
cAMP-CAP complex hardly occurs which produces a leaky expression. But during low levels of
glucose the cAMP raises and this cAMP-CAP complex binds and bends the DNA and allows the
RNA polymerase to bind to the promoter and transcribe the Lac structural genes.
Similar to the mechanism of alloctose, isopropylthiolgalactoside (IPTG) induces the
expression of the Lac operon, which binds to the repressor protein and changes its
conformational "fingers" that normally inhibits its binding to the operon. By using an inducer
medium to interfere with the pathways in parallel cultures we can then calculated the amounts
enzymatic activity of B-galactosidase metabolized in a cell by using a molecule called O-
nitrophenol galactoside.
O-nitrophenol galactoside(ONPG) is of substrate of B-Gal and when hydrolyzed the
molecule breaks into Galactose and O-nitrophenol(ONP), which turns yellow when a phosphate
buffer is added. The amount yellow indicates the amount of ONP, thus the expression of B-gal.
By getting different concentrations of ONP we can then plot points on a graph by obtaining the
absorbency reading of each concentration and calculating the Moles of ONP, we made five
different samples of ONP and phosphate buffer concentrations to make the points on the plot,
which are indicated in the results as the Standard Curve. We then incubated cultures at
different times to measure the Moles of ONP to find how much was being produced per second
and per cell, thus showing how much B-gal was produced from the Lac Operon genes.
Each pair of students made five test tubes containing different concentrations of ONP
and phosphate buffer with a total volume of 1.6 ml. Tube 1 contained 0ml of ONP and 1.6 ml of
phos. buffer, which was used as a blank for the spectropometer. The second tube have .16ml ,
third contained .32ml, fourth .48ml and fifth had .64 ml of ONP, the rest was filled with
phosphate buffer to get the 1.6 ml total. Each tube was then given 1.2ml of 1M sodium
carbonate and placed into the Spectrophotometer at an absorbance of A420 and the resulted
were taken. The volumes are identical to the volumes used for the procedure in the B-gal assay
so we can calculate the Moles of ONP using the standard curve in the results.
Five screw cap tubes were each given 2.5ml of Z buffer and 0.2 ml of toluene and
labeled 0, 4, 10, 25, 50 minutes, according to the amount of time left in the incubator later on
in the experiment. Five culture tubes were made each with the same labels. Each pair of
students received a flask containing a 5 ml culture in a 40 ml minimal medium which was
incubated overnight. Then 5 ml of the culture was given to the "0" culture tube and 3 ml of .1 M
of IPTG was added to the remaining 40 ml of culture and mixed well. As mentioned before
molecule inhibits the repressor protein for the lac operon, thus helping the promotion of the
lac structural genes.
The 40ml culture with the IPTG is then quickly distributed at 5 ml, aliquots of induced
cells, in to the remaining culture tubes, (4,10,25,50 min.) and is then taken to a shaking 37
o

water bath for the amount of time indicated on the label. After each tube is taken out 2.5 ml of
each incubated culture was then transferred to the screw cap tubes, respectively to their label,
which contained the toluene and Z buffer. This toluene is a chemical that kills the bacteria and
releases the B-galactosidase enzyme. The tubes are placed in another shaker water bath to full
lysis the cells.
Five test tubes were made with 1.3ml of 0.5% ONPG, labels with the same identification
as before. After the screw cap tubes were done in the water bath, 0.3ml aliquots of cells, under
the toluene layer, were transferred to each tube with the respective label and mixed. They
were placed into a 30
o
water bath until the ONPG separated and turned the mixture yellow
since the released B-gal is able to break the bond between ONP and galactose. After the
Amount of ONP Abs (420)
0 0
1.60E-07 0.193
3.20E-07 0.27
4.80E-07 0.546
6.40E-07 0.688
expected tubes turned yellow 1.2 ml of 1M sodium carbonate was added to stop the B-gal
enzymatic activity. Each Sample was transferred into a specto cuvette to take an Absorbance
reading at 420nm, the "0" tube was the blank to zero out the machine. The reading were
recorded and calculated using the standard curve from the first part of the experiment. A serial
dilution was made using the culture that was incubated at 50 minute before the toluene was
added to measure the growth, so we can calculated the point of the experiment which is the
amount of B-galatosidase made per second per cell.
Results
The Standard curve was made by using the different mole concentrations of ONP with
the phosphate buffer, and the absorbance of each of the five tubes where recorded in the chart
below. This chart was then graphed, giving us the Standard Curve and the equation so that we
can calculate the ITGP reaction later in the experiment. Table 1 shows the Absorbance of each
sample of different mole concentrations of ONP and Graph 1 is the plot for the Standard curve.

Table 1






Graph 1

After the absorbency readings were taken to later calculate the Standard curve and its
function, we then preformed the protocol of incubating five cultures to specific time limits
labeled to each tube, this way the cells can produce different amounts of B-galactisdase by the
IPTG inducer molecule. After the incubation period, each tube was then mixed with toluene and
Z-buffer to lysis the cells membrane which released the entire B-gala enzymes produced. ONPG
was then added to react with the B-gala to produce ONP, which turns a more intense yellow
color depending on the amount of B-Gala reacting with the ONPG. The intensity was then
measured by the Absorbency readings and recorded on the chart below, Table 2. These
numbers were then used with the equation given by the Standard curve to calculate the Mole
of ONP produced, which was then used to calculate the moles of ONP per second.
Standard curve equation
1E+06x + 0.0136 = Y, Y= Abs(420)
y = 1E+06x + 0.0136
R = 0.9973
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0E+00 1E-07 2E-07 3E-07 4E-07 5E-07 6E-07 7E-07
A
b
s

(
4
2
0
)

Moles of ONP
O-Nirtrophnolate Standard Curve
Series1
Linear (Series1)


= X (Moles of ONP)
, then


= Moles per second

Table 2
Tubes
(minutes of incubation)
Abs 420
Y
Moles of ONP
X
Moles per second
0 mins 0 1.36E-08 0
4 mins 0.022 8.40E-09 2.10E-09
10 mins 0.135 1.21E-07 1.21E-08
25 mins 0.721 7.07E-07 2.83E-08
50 mins 0.851 8.37E-07 1.67E-08

After we calculated the Mole per second, we then could find out how many moles per
second were made by each cell. From our last procedure we preformed and serial dilution on
the 50 minute incubated tube that wasnt lysed by toluene. We then plated 100ul of the last
two dilution tubes, 10
5
and 10
6
, we then incubated them and counted the cell growth on each
plate a couple of days after. Plate 10
5
had over 300 colonies, so we used the 10
6
dilution plate
,which had 41 colonies and was used to calculate our final equation and question of this
experiment, How many moles of ONP were produced each second by each cell?. The Moles
per second calculation, that was previously done above was divided by the number of cells in
the counted original culture, so we calculated the dilution factors of the serial dilution and the
dilution that made to make the 50 minute culture tube.
(The 2.5 ml of culture transfer into toluene is not counted to find the number of bacteria in
culture)



Number of cells
First dilution: 5ml of culture was transferred, diluted with 3ml IPTG

= .625 so DF= 1.6

Second Dilutions: Serial Dilution
4.1 x 10
6
Total Dilution
4.1 x 10
6
x 1.6 =
6.56 x 10
6
Cells in culture
Final calculations


=

2.545731 x 10
-15

Discussions

A cell using this kind of regulating mechanism, seen in the lac operon, is beneficial to
their growth and survival. When a cell needs a specific protein activated due to the lack or
stimulating environment, this kind of repression and activation causes the cells to only use
energy to produce specific enzymes and proteins when needed.
ONPG is a chemical that breaks into ONP and galactose when in the presents of B-gal,
which breaks the ester bond between these molecules. The amount of B-gal created by the cell
will cause this reaction to happen, so the more B-gal created means the more reactions are
taking place producing ONP which turns yellow. The more yellow the solution is the more ONP
was created by B-gal, so there is a direct correlation between ONP and B-gal. If the incubation
period was increased then more cells would be made, thus express and produce more B-gal,
this in turn would increase the moles of ONP per second, due to the excesses amount of B-gal
being produced by each cell.
Two of our induced culture tubes, at 25 and 50 minutes were over the highest known
points on the standard curve. This could lead to some inaccuracy in calculations since the linear
equation was fit for the points we found at different ONP concentrations, but if more readings
were taken at higher ONP levels then our linear equation would fit this setting. Since we can
assume the standard curve is close to straight line and our R value is close to 100%, we can
safely calculated higher points then on the standard curve, even though there may be slight
errors.