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Induction of -galactosidase in Eschericia Coli

Introduction The lactose operon in Escherichia coli is one of the rst 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 has been 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 for key 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].

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 the positive control mechanism of the lac operon.

Fig1: The Haworth structures for ONPG and IPTG.

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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.

Time of Induction (Min)

0 1 2 3 4 5 7 10 12 15 30 45 0c 15c

A414 (Average)
.062 .064 .072 .127 .139 .245 .301 .450 .672 .491 1.291 1.930 .071 .066

Corrected A414
0 .002 .01 .065 .077 .183 .239 .388 .610 .429 1.291 1.868 .009 .004

Units Galactosidase per ml of culture

0 .0000834 .000420 .00273 .00323 .00786 .0100 .0162 .0256 .0180 .0542 .0784 .000378 .000168

45c

.069

.007

.000294

Table 1: Time course induction of -galactosidase by IPTG.

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-nitrophenol per 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

Plot of Units of -galactosidase per ml of culture against Time of induction

Units of -Galactosidase per ml of culture.(mole/ml) 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 -0.01 0

10

20

30

40

50 IPTG Control

Induction time (Minutes)

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 Time of Induction IPTG B IPTG + Chloramphenicol Lactose C D IPTG 5mM + Glucose IPTG 10mM+ glucose E IPTG + rifampicin F G IPTG + Streptomycin

0 min (Blank) 5 min 10 min 15 min 30 min 45 min

0 .00502 .0141 .0210 .0470 .0539

0 .01302 .0257 .0311 .0283 .0201

0 .00268 .00625 .0092 .00187 .0193

0 .00193 .00457 .00735 .0282 .0298

0 .00281 .00462 .00806 .0185 .0240

0 .01062 .0219 .0300 .0338 .0330

0 .0068 .0184 .0246 .0254 .0243

Table 2: Collated Laboratory group results for Part B Characteristics of the Induction

Units of -galactosidase per mL of culture (mole/mL)

0.06 0.05 0.04 0.03 0.02 0.01 0 0 10 20 30 40 50 Time of Induction (Minutes) IPTG IPTG + Chloramphenicol Lactose IPTG '5mM' + Glucose IPTG '10mM' + Glucose IPTG + rifampicin IPTG + Sreptomycin

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 pipette technique 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

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 onitrophenol. 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.coli cells, 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

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

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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 (5th 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.

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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