Bioc Module 2 Lab Report
Bioc Module 2 Lab Report
Bioc Module 2 Lab Report
BIOC 221
Declaration Sheet for Individual Assignment
Lab report: Module 2
Full Name:………Mimi Boister…………………………………………………
Student ID:…………6305238……………………………………………..
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The lac operon is also regulated by the presence or absence of glucose. If there are high
glucose levels in the cell, this means that glucose gets used up in glycolysis to produce ATP
and therefore cyclic AMP (cAMP) levels decrease (Nature Publishing Group, 2014). This
means that cAMP will not bind to the CRP receptor and the lac operon will not be activated.
When glucose is not present, cAMP does bind, and the lac operon is expressed. This system
is known as catabolite repression.
This experiment aims to compare the effects of the antibiotics Chloramphenicol and
Rifampicin on β-galactosidase activity in relation to the lac operon. This analysis is intended
to either reinforce or disprove the accepted theory of the lac operon, that an increase in β-
galactosidase activity occurs due to increased de novo synthesis of the protein.
BIOC221 – Module 2 Lab Report – Mimi Boister - 6305238
In this investigation an enzyme activity assay was completed, which involved various
experimental techniques.
The first of these techniques was the lysis of cells. This involved intermittently centrifuging
and vortexing the culture samples of bacterial cells to break the bacterial cell walls and
access the β-galactosidase in the cell.
The second technique used in this enzyme assay was the activity assay itself. This consisted
of adding ONPG (ortho-Nitrophenyl-β-galactoside) to a test tube, vortexing to mix, and
starting to time, before incubating at 37 degrees Celsius until a visible amount of yellow
appeared (this yellow is due to ONP, the product). Then the absorbances of each sample in
the test tubes were taken, to determine the amount of product formed after the time
period and therefore the level of activity of the β-galactosidase (as activity refers to the
efficiency of the enzyme’s action) (BIOC221 teaching staff, 2023). The conversion of ONPG
to ONP is catalysed by β-galactosidase, therefore the amount of product (ONP) corresponds
to the enzyme units/mL
To measure the amount of product formed in the assay, and therefore the enzyme activity,
a standard curve first had to be constructed for ONP. The specific volumes for each sample
in Table 3 of the appendix were used. Each sample was then put into a cuvette and then into
a spectrophotometer, which measures the absorbance. The spectrophotometer works by
measuring how much light is transmitted, therefore how much has been absorbed by solute
molecules, to find the concentration of the solution. There is a linear relationship between
absorbance and concentration which allows the standard curve to be formed. This standard
curve could then be used as a reference to determine the amount of ONP liberated at
different time points after induction for this experiment, and therefore the amount of β-
galactosidase.
Method
Before beginning the enzyme assay or cell density determination, the standard curve was
constructed using the volumes from Table 3 in the appendix and following the instructions
on page 2-16 of the lab manual under the subheading “Standard curve”.
Once the demonstrators had obtained enough cells by waiting for the bacteria to enter the
growth phase (approx. 0.3 absorbance reading), steps 4 and 5 of the method on page 2-13
were completed. 0.5 mL of the 2 mL sample of the E. coli culture was then used for the lysis
of cells and the activity assay on page 2-15, and the remaining 1.5 mL portion was used for
the determination of cell density on page 2-16.
BIOC221 – Module 2 Lab Report – Mimi Boister - 6305238
Another 2 mL sample was taken from the flask 20 minutes after the first sample and steps 6
and 7 of page 2-13 were followed. While this was taking place, 3 additional flasks were also
being labelled to prepare for the antibiotics to be added into the experiment.
At t=40, yet another 2 mL sample was taken for β-galactosidase and the cell density to be
determined (p2-16, 2-17). However, at this step there was variation from the method in the
lab manual. A 5 mL pipette was used to administer 10 mL of culture from the original flask
into the three newly labelled flasks. One flask, labelled “original”, was not treated. This was
used as the positive control to ensure that the IPTG was working as the inducer of the lac
operon and there were no external factors affecting the experiment.
In terms of the remaining flasks, one labelled “Rifampicin” had 150 microlitres of the 10
mg/mL Rifampicin antibiotic added to it using a 2/200 micropipette, while the other that
was labelled “Chloramphenicol” had 10 microlitres of the 20 mg/mL Chloramphenicol
antibiotic added to it (see materials on pg 2-12 of lab manual). These three flasks were then
added back into the shaker. The volumes of Chloramphenicol and Rifampicin to add were
determined using their respective concentrations and the 10 microlitre volume in the flask,
by the c1V1 = c2V2 relationship (BIOC221 teaching staff, 2023).
At t=60 and t=80, 2mL samples were taken from each of the three flasks and the β-
galactosidase assay and cell density determination were carried out as above for each
sample.
Results
Table 1. Enzyme units β-galactosidase per mL: Table showing the amount of ONP liberated
(and therefore enzyme units per mL) at each time point after β-galactosidase induction for
the original, Rifampicin treated, and Chloramphenicol treated E.coli samples. 1 enzyme unit
liberates 1 mol of o-nitrophenol in 1 min.
(mmol.mL-
1
)
Assay 60 7.75 6 8.5 2.5 3.25 2.75 4.25 3.75
period
(min)
ONP 0.00333 0.11613 0.21667 0.17059 0.38 0.277 0.291 0.165 0.242
liberated x2=
mmol x 0.34118
mL-1
= Enzyme
Units.mL-1
Table 1 shows the amount of ONP liberated (and therefore enzyme units/mL) at each time
point after β-galactosidase induction for the positive control (as shown by “original”), the
flask with Rifampicin added (shown by the subscript “R” at 60 and 80 minutes) and the flask
with Chloramphenicol added (shown by the subscript “C” at 60 and 80 minutes)
respectively. The positive control shows a linear increase in ONP liberated as the time after
induction increased. For t=60, a 1 in 2 dilution of the solution was carried out because it was
left too long to change to a yellow colour and had too high of an absorbance reading.
Therefore, this dilution had to be accounted for by multiplying the ONP liberated by two to
obtain the true value. The Rifampicin culture shows a value of 0.277 enzyme units/mL for 60
minutes after induction, which is an increase from the value for the 40-minute time point
for the control. For this culture, the enzyme units/mL continued to increase between the
time points of 60 minutes and 80 minutes. Finally, the Chloramphenicol shows a value of
0.165 enzyme units/mL for t=60, which is a decrease from the 40-minute time point for the
control. The enzyme units/mL for the culture with Chloramphenicol then increased again
between 60 and 80 minutes as shown by the last column of the table.
Table 2. Enzyme Units of β-galactosidase per E coli cell – Table showing the enzyme units
per E. coli cell at each time point after β-galactosidase induction for the original, Rifampicin
treated and Chloramphenicol treated E. coli samples.
Table 2 shows the enzyme units of β-galactosidase per cell at each time point for the
positive control flask (as shown by “original”), the flask treated with Rifampicin (as shown by
the subscript “R”) and the flask treated with Chloramphenicol (as shown by the subscript
BIOC221 – Module 2 Lab Report – Mimi Boister - 6305238
“C”) respectively. The original culture shows a steady increase in the enzyme units/cell,
however there is a slight decrease between the time points of 60 and 80 minutes. The
Rifampicin culture shows a slight increase from the t=40 time point for the positive control
(from 2.183 x 10-10 to 2.67 x 10-10). The Chloramphenicol culture shows a decrease and then a
slight increase from the t=40 time point for the positive control (1.43 x 10-10, then 2.23 x 10-10).
From this table, one can also see the cells/mL for the various samples against time after
induction. For the original culture there is a continuous increase in the cells/mL, whereas for
the Rifampicin and Chloramphenicol cultures the cells/mL remains rather stationary from 40
minutes to 80 minutes after induction.
4.00E-10
3.00E-10
Enzyme units/cell
2.00E-10
1.00E-10
5.17E-26
0 20 40 60 80
-1.00E-10
Figure 1. Enzyme Units of β-galactosidase per E. coli cell against Time after Induction. A
graph showing the trends in the enzyme units of β-galactosidase per cell every 20 minutes
after induction. The blue line represents the enzyme units/cell for the original culture of
E.coli cells, the orange line represents the enzyme units/cell for the culture treated with the
antibiotic Rifampicin, and the grey line represents the enzyme units/cell for the culture
treated with the antibiotic Chloramphenicol. The orange and grey lines begin at 40 minutes
as this is when the antibiotics were added to the separate flasks of E.coli culture.
Looking at the graph, there is a steady increase in the enzyme units/cell for the original
culture until t=60, after which the enzyme units/cell decreased slightly below those of the
Rifampicin culture. There is a slight increase in enzyme units/cell above the original culture
BIOC221 – Module 2 Lab Report – Mimi Boister - 6305238
for the Rifampicin culture. Finally, there is a decrease in the enzyme units/cell for the
Chloramphenicol culture from t=40 to t=60, followed by an increase to t=80.
In terms of the Rifampicin sample, a slight lag in the enzyme units/cell was expected
between 40 and 60 minutes after induction and then a plateau between 60 and 80 minutes.
This lag and then plateau in the enzyme units/cell would be due to Rifampicin acting as an
inhibitor of transcription in the E. coli cells. Rifampicin achieves this by binding to the
catalytic β-subunit of RNA polymerase II (Brown, 2023). This inhibits the action of RNA Pol II
and therefore the DNA in the lac operon, particularly the lacZ gene, would be unable to be
transcribed, meaning no production of β-galactosidase. However, there would already be
some mRNA transcripts present in the cell, meaning these could be translated to produce
the β-galactosidase enzyme, hence why there would not be a complete halt in enzyme
activity but a lag instead. After 60 minutes the Rifampicin sample enzyme units/cell should
have begun to plateau as the mRNA ran out and no transcripts were further produced. The
reason for this plateau would be due to there being no growth of the cells (as shown by the
halted cells/mL Rifampicin curve in Figure 3 of the appendix). This combined with the long
half-life of β-galactosidase as mentioned previously would mean the same amount of β-
galactosidase protein would be present in the cells, resulting in the same enzyme units/cell
values. The results from this experiment showed a continued increase in the enzyme
units/cell with Rifampicin from 40 minutes onwards. This increase cannot be attributed
solely to the presence of mRNA transcripts in the cell, as the half-life of mRNA in E. coli cells
BIOC221 – Module 2 Lab Report – Mimi Boister - 6305238
is generally between 3-8 mins (Aiba, 2002), not enough time for β-galactosidase activity to
still be occurring as shown in the graph and tables. There could have been many reasons for
this outcome, one of them being that too low of a volume of Rifampicin was added to the
flask by accident. This would have decreased the effect of the antibiotic on RNA polymerase
II and the binding of the β-subunit, allowing a greater extent of transcription to occur than
with a higher volume and resulting in a continued increase in the enzyme units/cell
following the control curve. However, potentially a more likely reason for this perceived
increase is due to the control being an inaccurate reference for β-galactosidase units/cell. If
the control had not plateaued between 60 and 80 the relative comparison would have
shown that Rifampicin did slightly decrease the enzyme units/cell and was working as
expected.
The results with the antibiotic Chloramphenicol added showed a clear decrease in the
enzyme units/cell between 40 and 60 minutes, followed by an increase between 60 and 80
minutes after induction. A plateau in the enzyme units/cell was expected for
Chloramphenicol as the antibiotic inhibits cell growth, as shown by the stagnancy of the
cells/mL curve in Figure 3, but the long half-life of β-galactosidase results in the enzyme
units/cell being stationary. Regardless, the decrease in enzyme units/cell reinforces the idea
that Chloramphenicol is an inhibitor of translation. The antibiotic specifically inhibits the
elongation stage of translation, as it binds to adenines at the peptidyl transferase centre of
ribosomal RNA. This means that the ribosome can no longer act as a catalyst for peptide
bond formation, and no protein can be produced (Legerwood, 2023). Therefore, with
Chloramphenicol present, no further β-galactosidase should be produced, and the enzyme
units/cell should plateau or decrease (Sypherd and Strauss, 1963). This decrease also
demonstrates the fact that the increase in β-galactosidase activity is due to de novo protein
synthesis, rather than another factor influencing enzyme activity. This is because when
translation (protein synthesis) is being inhibited but there is the same number of cells, as
shown by the Chloramphenicol curve in the cells/mL graph of the appendix, the β-
galactosidase activity decreases. Therefore, the Chloramphenicol inhibition shows that any
increase in β-galactosidase activity for the control is due to the induction of the lac operon
rather than an increase in the number of cells within the culture. Subsequently, the increase
in enzyme units/cell between 60 and 80 minutes after induction was unexpected. Again,
there could have been many reasons for this, including that a volume smaller than 10 l was
added, or the Chloramphenicol used was not the correct concentration, reducing the effect
of the inhibitor. There is little evidence to suggest any particular factors that could have
caused the Chloramphenicol to stop working properly, thus the increase in enzyme
units/cell is likely to be due to an experimental error, such as those listed above.
To conclude, the results of this experiment were not successfully able to show the effect of
the antibiotic Rifampicin on the lac operon. However, the results did partially show the
effect of Chloramphenicol and therefore the concept of de novo synthesis of β-galactosidase
after being induced by IPTG. Therefore, while the results were somewhat unexpected, there
were a few critical conclusions that could be made from this investigation.
References
Aiba, H. (2002). Faculty opinions recommendation of global analysis of mrna decay and
abundance in escherichia coli at single-gene resolution using two-color fluorescent DNA
BIOC221 – Module 2 Lab Report – Mimi Boister - 6305238
Brown, C (2023) How does bacterial transcription start? – how can we inhibit it?, slides 8-13,
BIOC 221 Molecular Biology, Biochemistry, University of Otago, Dunedin.
Brown, C (2023) Transcription Termination and the Regulation of transcription, slides 12-18,
BIOC 221 Molecular Biology, Biochemistry, University of Otago, Dunedin.
BIOC221 teaching staff (2023) Control of gene expression. In BIOC 221: Molecular Biology
2023. pp 2.1. – 2.52, Biochemistry, University of Otago, Dunedin.
Hediger, M. A., Johnson, D. F., Nierlich, D. P., & Zabin, I. (1985). DNA sequence of the lactose
operon: The laca gene and the transcriptional termination region. Proceedings of the
National Academy of Sciences, 82(19), 6414–6418. https://doi.org/10.1073/pnas.82.19.6414
Saqib, S., Akram, A., Halim, S. A., & Tassaduq, R. (2017). Sources of β-galactosidase and its
applications in food industry. 3 Biotech, 7(1). https://doi.org/10.1007/s13205-017-0645-5
Warmerdam, A., Boom, R. M., & Janssen, A. E. (2013). Β-galactosidase stability at high
substrate concentrations. SpringerPlus, 2(1). https://doi.org/10.1186/2193-1801-2-402
Appendix
Tube 1 2 3 4 5 6
(blank)
o-Nitrophenol (2mmolL-1) 0 0.05 0.10 0.15 0.20 0.25
Sodium phosphate (mL) 5.2 5.2 5.2 5.2 5.2 5.2
Sodium carbonate (mL) 2.0 2.0 2.0 2.0 2.0 2.0
Water (mL) 0.8 0.75 0.7 0.65 0.6 0.55
Total Volume (mL) 8.0 8.0 8.0 8.0 8.0 8.0
A420 0.000 0.188 0.357 0.554 0.6925 0.879
ONP (mol) 0.0 0.1 0.2 0.3 0.4 0.5
Standard curve
0.4
0.3
0.2
0.1
0
0 0.1 0.2 0.3 0.4 0.5 0.6
ONP (μmol)
Figure 2. The standard curve of A420 against ONP (mol). This curve was obtained using the
values in Table 3, to discover the absorbances at increasing amounts of ONP, which is the
product of the hydrolysis of ONPG (ortho-Nitrophenyl-β-galactoside) by β-galactosidase.
This reaction allows us to use the standard curve as a reference to measure β-galactosidase
activity at different time points. The curve is linear because the relationship between
absorbance and concentration is linear.
BIOC221 – Module 2 Lab Report – Mimi Boister - 6305238
1.60E+09
1.40E+09
1.20E+09
1.00E+09
Cells/mL
8.00E+08
6.00E+08
4.00E+08
2.00E+08
0.00E+00
0 20 40 60 80
Figure 3. The number of E.coli cells/mL against time after induction. The blue line represents
the cells/mL for the sample with no antibiotics added. The orange line represents the
cells/mL for the sample with Rifampicin added. The grey line represents the cells/mL for the
sample with Chloramphenicol added.