Induction of β
-
Upload
vaibhav-bhandari -
Category
Documents
-
view
214 -
download
0
Transcript of Induction of β
-
8/3/2019 Induction of
1/11
1
Induction of -galactosidase in Eschericia Coli
Introduction
The lactose operon in Escherichia coli is one of the first and most studied examples
of gene regulation and control in prokaryotes. The lac operon regulates the production
of enzymes involved in the metabolism of lactose, and incorporates both positive and
negative feedback in its regulatory network .The aim of this experiment is to study IPTG
induced production of -galactosidase , a hydrolase enzyme, to illustrate gene control
as predicted by simple models of the system. An assay of -galactosidase activity hasbeen conducted as part of this experiment.
Many protein-coding genes in bacteria are clustered together in operons which serve
as transcriptional units that are co-coordinately regulated. One of the most studied of
these is the lac operon in E.coli. The operon model proposes three elements, a set of
structural genes along with an operator and promoter site. In the lac operon , the
structural genes are the lacZ, lacY and lacA genes encoding -galactosidase , -
galactoside permease and thiogalactoside transacetylase [6]. This operon codes forkey enzymes involved in lactose metabolism and as lactose availability increases in
E.coli cells there is a substantial and co-ordinated increase in the amount of each
enzyme. Thus each enzyme is known as an inducible enzyme and the process is called
induction.
In this experiment however, isopropylthiogalactosidase (IPTG) functions as a mimic
inducer molecule instead of the natural allolactose due to its ability to not be
metabolized by E.coli . During induction, the inducer molecule binds to the repressor
that greatly reduces its affinity for the lac operator site. The dissociation of the lac
repressor from the operator site allows the RNA polymerase to begin transcribing the
lacZ lacY and lac A genes which in turn leads to many copies of the polycistronic mRNA
and after translation, large amounts of all three enzymes [1].
-
8/3/2019 Induction of
2/11
2
In this bacterium, glucose is the preferred and most commonly used source of energy,
but lactose is an alternative when glucose is not available. The lactose operon which is
a small cluster of genes including the one for -galactosidase , functions in such a way
that the enzyme is synthesized only when lactose is present . This is achieved through
an efficient feedback mechanism where lactose is converted to allolactose , which is
capable of attaching to the repressor and prevent binding to the operator site. The RNA
polymerase now binds to its site and synthesizes -galactosidase enzyme which now
selectively hydrolyses allolactose, decreasing the amount of inducer in the cell and
reducing -galactosidase expression by virtue of a negative feedback mechanism. By
the use of an inducer such as IPTG (isopropyl--thiogalactopyranoside), the lac
repressor is no longer able to act on the operator and transcription is de-repressed
[1,5].
In the following experiment, we have used the lactose analogue isopropyl
thiogalactoside (IPTG) to induce lac operon expression in E. coli. IPTG is easily
transported into normal E. coli cells where it binds and inhibits the action of the LacI
repressor protein and effectively activates lac operon expression. Unlike lactose though,
IPTG cannot be cleaved by -galactosidase and remains within the cell as a constant
activator of lac operon expression [9]. O-nitrophenyl -galactoside (ONPG) is another
lactose analogue which in this experiment we aim to use quantitatively, to measure the
enzyme activity of -galactosidase. ONPG is a colourless substrate that can be cleaved
by the enzyme -galactosidase to yield stoichiometric amounts of yellow o-nitrophenol
and colourless galactose. As yellow o-nitrophenol is produced, its concentration can be
measured by reading absorbance at 414nm. The characteristic effects of glucose ,
chloramphenicol ,rifampicin and stretptomycin on -galactosidase induction have also
been investigated .The inability of E.coli to produce lactose utilization enzymes in the
presence of an alternate substrate such as glucose have been studied as part of thepositive control mechanism of the lac operon.
-
8/3/2019 Induction of
3/11
3
Fig1: The Haworth structures for ONPG and IPTG.
[Original source- http://www.pearsonhighered.com]
Materials and Methods
The experimental procedure conducted is documented in detail in the laboratory
manual for BIOC2201 [5]. It is to be noted that for Part B: Characteristics of the
induction of -galactosidase, similar procedure to Part A was employed in addition to
the selected variations.
Alternate V variation:
1. 250 l IPTG (5mM) + 250l glucose (20mM)
2. 250 l IPTG (10mM) + 250l glucose (20mM)
Results
The following results were the result of experimentation in the BIOC2201 laboratory.
Results from Part B were compiled with assistance from other students in the laboratory
group who performed different variations.
http://www.pearsonhighered.com/http://www.pearsonhighered.com/ -
8/3/2019 Induction of
4/11
4
Time of
Induction(Min)
A414
(Average)
Corrected A414 Units -
Galactosidaseper ml of culture
0 .062 0 0
1 .064 .002 .0000834
2 .072 .01 .000420
3 .127 .065 .00273
4 .139 .077 .00323
5 .245 .183 .00786
7 .301 .239 .0100
10 .450 .388 .0162
12 .672 .610 .0256
15 .491 .429 .0180
30 1.291 1.291 .0542
45 1.930 1.868 .0784
0c .071 .009 .000378
15c .066 .004 .000168
45c .069 .007 .000294
Table 1: Time course induction of -galactosidase by IPTG.
-
8/3/2019 Induction of
5/11
5
Calculations
Given values [5]:
414 of ONPG = 21300 M-1cm-1
Path length = 0.9 cm
Assay volume 0.8 ml
At t = 1 minute,
Corrected A414 = .002
Using the Beer Lambert law,
A = cl
Where we take A as absorbance, C as concentration and l as pathlength.
C =A/ ( X l)
.002 /(21300 x 0.9)
1.0433 x 10-6
1.0433 M
By taking 0.8ml assay volume and calculating C,
C = 1.0433 mole x 0.8ml / 1000ml
8.346 x 10-4 mole
Given that one unit of galactosidase is the amount of enzyme that will
catalyse hydrolysis of 1mole of ONPG to o-nitrophenolper minute. The sample
was incubated for 5 minutes after the addition of ONPG and 200l of E.coli
sample was used.
Units of galactosidase formed / minute = 8.346 x 10-4mole / 5
Units of of galactosidase formed /minute/ml = 8.346 x 10-4mole / 5 x 0.2
8.346 x 10-4mole min-1 ml-1
-
8/3/2019 Induction of
6/11
6
1: Graph of Units of -galactosidase per ml of culture against Time of induction
The induction time for the lac operon as determined from the above graph is 5 minutes
to 6 minutes. This result is consistent with the model data set provided when IPTG is
employed as an inducer [5].
Condition
A B C D E F G
Time ofInduction
IPTG IPTG +Chloramphenicol
Lactose IPTG 5mM+ Glucose
IPTG10mM+glucose
IPTG +rifampicin
IPTG +Streptomycin
0 min(Blank)
0 0 0 0 0 0 0
5 min .00502 .01302 .00268 .00193 .00281 .01062 .0068
10 min .0141 .0257 .00625 .00457 .00462 .0219 .0184
15 min .0210 .0311 .0092 .00735 .00806 .0300 .0246
30 min .0470 .0283 .00187 .0282 .0185 .0338 .0254
45 min .0539 .0201 .0193 .0298 .0240 .0330 .0243
-0.01
0
0.01
0.02
0.03
0.04
0.050.06
0.07
0.08
0.09
0 10 20 30 40 50
Unitsof-Galactosidasepermlof
culture.(
mole/ml)
Induction time (Minutes)
Plot of Units of -galactosidase per ml of culture against Time ofinduction
IPTG
Control
-
8/3/2019 Induction of
7/11
7
Table 2: Collated Laboratory group results for Part B Characteristics of the Induction
2: Graph depicting the Units of -galactosidase per ml of Bacterial culture versus the Induction
time (Raw data obtained collectively by Laboratory group).
Discussion
Our examination of the kinetics of -galactosidase induction in part A of the
experiment suggests that enzyme production began increasing 5-6 minutes after the
addition of the inducer (IPTG) AT 37C (Graph 1). It was found that Units of -
galactosidase enzyme had an increasing pattern over the course of the induction after
the initial lag phase. However, the results showed a deviation from the model data set at
time point 12 minutes and 15 minutes. This can only be attributed to inaccurate pipettetechnique as care was taken to maintain the samples at the required 37C at all times.
A steady state rate of enzyme synthesis after induction can be observed if the
inaccuracies of the two data points is ignored whereas the time required to reach this
steady-state rate are dependent on IPTG concentration. The rate of enzyme synthesis
and the time constant for induction are known to show quite different dependences on
0
0.01
0.02
0.03
0.04
0.05
0.06
0 10 20 30 40 50Unitsof-galactosidasepermL
ofculture(mole/mL)
Time of Induction (Minutes)
IPTG
IPTG + Chloramphenicol
Lactose
IPTG '5mM' + Glucose
IPTG '10mM' + Glucose
IPTG + rifampicin
IPTG + Sreptomycin
-
8/3/2019 Induction of
8/11
8
the kind of inducer employed and on the concentration of these inducers [2]. In our
experimentation it is not entirely evident whether the induction of the lac operon was
instantaneous, however we did not note an absorbance level above zero for 0 time point
samples from both the Induction and control flasks.
In this experiment the addition of CTAB (cetyl trimethyl ammonium bromide) functions
as a bactericide causing the E.coli cells to lyse and release -galactosidase. The -
galactosidase then hydrolyses the ONPG (O-nitrophenyl -galactoside) to yield o-
nitrophenol. The amount of o-nitrophenol formed can be measured by determining the
absorbance at 420 nm. The amount of o-nitrophenol produced is proportional to the
amount of -galactosidase and the time of the reaction. In addition, Na2CO3 is
employed to stop the reaction by shifting the reaction mixture to pH 11 [2]. At this pH
most of the o-nitrophenol is converted to the yellow colored anionic form and -
galactosidase is inactivated [3,5].
From the analysis of (Graph 2) the results for Part B of the experiment we can deduce
that the induction by IPTG is more effective than that of lactose. As lactose needs to be
converted to allolactose by the E.colicells, less of it remains available for use as it is
hydrolyzed by -galactosidase. There is an apparent reduction in the concentration of
the natural inducer allolactose in this case and it causes a reduction in the effectiveness
of the induction. In comparison, IPTG is not metabolized by -galactosidase and is
maintained at the initial concentration in the cell causing de-repression of the lac operon
to be sustained and gene expression activated. It is also known that the lack of negative
feedback mechanism when IPTG is used, would allow the inducer to accumulate to
greater concentrations than is the case for the natural inducer, allolactose. However this
variable was not tested in our experimental design.
The effect of IPTG and glucose, together, on the expression of the lacz gene is greatly
influenced by the feed-back mechanisms of the lac operon. As glucose represses -
galactosidase expression, the Catabolite activator protein (CAP) is needed to transcribe
the -galactosidase gene by facilitating the binding of RNA polymerase to the Lac
-
8/3/2019 Induction of
9/11
9
operon. CAP, in turn, relies on a physical interaction with cAMP in order to bind to the
Lac operon and increase the availability of RNA polymerase [5]. Since the levels of
cAMP are inversely proportional to the levels of glucose, the presence of glucose will
decrease the intracellular levels of cAMP, and diminish the ability of CAP to facilitate -
galactosidase transcription. In the presence of glucose and IPTG together, a basal level
expression of -galactosidase should be observed as transcription would be inefficient
due to the lack of RNA polymerase binding [2,5]. The results from our experiment do not
however accurately depict this basal expression in the samples containing IPTG and
glucose. Units of -galactosidase are much higher than expected values and this can
only be attributed to experimental error during the procedure.
The effects of three inhibitors of protein synthesis were also studied as part of this
experiment. Chloramphenicol and streptomycin are known to selectively inhibit
translation in bacteria whereas Rifampicin inhibits transcription. Chloramphenicol binds
to the 50s ribosomal subunit in bacteria and Inhibits transpeptidation (catalyzed by
peptidyl transferase). It blocks the binding of aminoacyl moiety of tRNA to mRNA
complex and as a result the peptide at the donor site cannot be transferred to the
amino acid acceptor [4] . It is a bacteriostatic inhibitor of protein synthesis.
Streptomycin binds to the small 16S rRNA of the 30S subunit of the bacterial
ribosome, These aminoglycosides irreversibly bind to the 30S ribosome and freeze the
30S initiation complex (30S-mRNA-tRNA) so that no further initiation can occur. This
leads to codon misreading, eventual inhibition of protein synthesis and ultimately death
of microbial cells. The aminoglycosides also slow down protein synthesis that has
already initiated and induce misreading of the mRNA. It is a bactericidal inhibitor of
protein synthesis. Rifampicin is also a bactericidal inhibitor of protein synthesis , which
binds to DNA-dependent RNA polymerase on the subunit and inhibit RNA synthesis
[4].
The mechanism of inhibitions possessed by these inhibitors can be confirmed by the
results of our experiment. The samples treated with the inhibitors exhibited the effects of
restricted induction but these results do not match the model data set due to
inconsistencies while experimentation. The inhibitory action on transcription and
-
8/3/2019 Induction of
10/11
10
translation is evident from the model data set of this experiment and can be
substantiated as being essential for the induction of -galactosidase. In this experiment,
evidence has been presented which suggests that the lac operon can be effectively
induced to synthesize -galactosidase in the presence of an artificial inducer IPTG. This
operon is also suitably inhibited by the three inhibitors of protein synthesis, which have
varying mechanism of action on transcription and translation. It can also be said that
glucose can affect the rate of expression of the lac operon by at least two distinct
mechanisms, either by reducing the internal concentration of inducers of the lac operon
or by catabolite repression of the lac operon.
References
1. Alberts , B .et al., Molecular Biology of the Cell (5 th Edition , 2008), Garland
science.
2. Boezi, J. A., and Cowi, D. B., Biophysical Journal. 1961, 1- 639.
3. Cohn, M., and K. Horibata. 1959. Physiology of the inhibition by glucose of the
induced synthesis of the, -galactoside-enzyme system in Escherichia coli. J.
Bacteriol. 78:624-631.
4. Conte ,John E (1994). Manual of antibiotics and infectious diseases: treatment
and prevention. Philadelphia: Lippincott Williams and Wilkins. 213-250.
5. Jacob, F. and J. Monod (1961). Genetic regulatory mechanisms in the synthesis
of proteins. Journal of Molecular Biology 3: 318-56.
-
8/3/2019 Induction of
11/11
11
6. Loomis, W. F., JR., and B. Magasanik. 1965. Genetic control of catabolite
repression of the lac operon in Escherichia coli. Biochem. Biophys. Res.
Commun. 20:230-234.
7. Lederberg, J. 1948 Gene control of -galactosidase in Escherichia coli. Genetics,
33,617-618.
8. Pardee, A. B., F. Jacob and J. Monod (1959). The Genetic Control and
Cytoplasmic Expression of "Inducibility" in the Synthesis of Beta-Galactosidase
by E. coli. Journal of Molecular Biology 1: 165-178