Experiment 5: Enzyme synthesis regulation in Escherichia coli

Objective: The aims of this experiment are to study the enzyme synthesis regulation in Escherichia coli cells and determine which substance is the inducer for enzyme synthesis.

Introduction: There are three proteins: LacZ--galactosidase, LacY-the lactose permease, and LacA-lactose transacetylase are encoded by the lactose operon. The LacI repressor gene that close to lac operon represses the operon and the presence of an inducer will inactivate the repressor. Although lactose is not an inducer, but it can indirectly induces the lac operon after a small amount has been isomerized to allolactose. This is an isomer of lactose, generated in a side reaction by the low basal levels of -galactosidase which are found before induction. IPTG (isopropyl-thio--D-galactosidase) is often used as inducer. IPTG is not metabolized and is of no use to cell as it is a gratuitous inducer. The use of less favoured substrates such as lactose is prevented when favoured carbon source such as glucose presents. Catabolite repression depends largely on the intracellular level of cyclic AMP. Catabolite Activator Protein (CAP) binds with cyclic AMP and the complex is known as cyclic AMP receptor protein (CRP). The level of CRP is constant. Transcription of catabolite sensitive operons such as the lac operon requires binding of CRPcAMP complex to the promoter region. Thus, RNA polymerase is able to bind to and transcribe the operon. The regulation of cyclic AMP levels is due mostly to changes in activity of adenylate cyclase which catalyses the conversion of ATP to cyclic AMP plus inorganic pyrophosphate. The presence of glucose will cause a drop in the activity of adenylate cyclase and hence there will be a drop in cyclic AMP levels. Glucose must be transported for this to happen, but it does not need to be broken down and metabolized. Nonmetabolizable analogs of glucose, such as 2-deoxyglucose, cannot be degraded but can be transported and also cause catabolite repression.

Materials: The materials involved in this experiment are E.coli(Lac+) culture, 0.002M lactose, 0.002M glucose, 0.002M IPTG, 0.002M PBG, 1.0mg/ml sodium deoxycholate, Toluene, 0.01M ONPG, 0.01M sodium phosphate buffer(pH7), and 2M sodium carbonate. The apparatus used are 37oC water bath shaker, sterile capped test tubes, ice bath, spectrophotometer, cuvettes and micropipettes.

Procedures: A. Cell Growth The cell growth is performed by lab officers.

B. Induction of Enzyme 4ml of starved E.coli cells (1x107cell/ml) was added into four large sizes (18mm) labeled test tubes respectively. Then, 0.2ml of 0.002M inducer, which are LAC, GLU, IPTG and dH20 were added in four labelled tubes respectively. Then each tube was closed with a cap, and they were placed in a 37oC water bath and shaken for 30 minutes.

C. Assay for Enzyme

1. Disruption of selective permeability Each 4.2ml of an induced E.coli culture was added with 50L of sodium deoxycholate(1.0mg/ml) and 50L of toluene. Then they were capped and placed in a 37oC water bath and shaken for 10 minutes. They were kept in ice bucket until needed for assays. 2. Enzyme assays 2.0 ml of 0.1M sodium phosphate buffer (pH7), 2.0ml lyses E.coli preparation and 0.2ml of 0.01M ONPG were added into each small labelled culture tube. Then these tubes were incubated for 15 minutes at 37oC without shaking. The reaction was stopped by adding 0.5ml of 2M sodium carbonate. Then the absorbance was read at 420nm in a spectrophotometer. The results were recorded.

Results: Labeled culture tube LAC GLU IPTG dH20 Amount of Absorbance(A) 1.783 -0.055 0.883 0 Presence of yellow colour in mixture Yes No Yes No

Discussion: IPTG is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon in this experiment. Its sulfur atom wills form a chemical bond which is non-hydrolyzable by the cell and preventing the cell to degrade the inductant. Consequently, the level of IPTG will not drop in cells. IPTG induces the transcription of the gene coding for beta-galactosidase. Beta-galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of beta-glycosides into monosaccharides. Allolactose is isomer of lactose and it acts as inducer for lac operon. The difference between lactose and allolactose is lactose is galactose-(1, 4)-glucose whereas allolactose is galactose-(1, 6)-glucose. In cells, lactose will be converted to allolactose by -galactosidase in a different reaction to the hydrolytic one. According to a study about this experiment, a null mutant of lac Z can still generate LacY permease when grown with IPTG but not when grown with lactose. This depicts the production of inducer inside the cells requires the conversion of lactose to allolactose. In the disruption of selective permeability part, the sodium deoxycholate and toluene are used. Their role is to destroy the selective permeability of the cell membrane and to permits the rapid cell entrance of ONPG compound. Then, ONPG compound will detect the presence of beta-galactosidase activity. The compound is usually colourless but it will become yellow colour when the -galactosidase hydrolyzes it into galactose and orthonitrophenol. Due to -galactosidase is required for lactose utilization, therefore the intensity of the colour produced can be used as a measure of the enzymatic rate. From the results, the highest absorbance value is the culture labelled LAC, followed by the culture labelled IPTG and culture labelled GLU. The culture labelled dH2O has zero absorbance value as it was read as blank. Based on the mechanism, there will be no negative absorbance value which the culture labelled GLU had shown. The culture labelled GLU should have absorbance value approximate to zero because the glucose will not induce the enzyme synthesis. On the other hand, culture labelled IPTG should be having higher absorbance value than culture labelled LAC. According to the mechanism, the lactose needs to convert to allolactose to induce the enzyme synthesis; however the IPTG does not need to undergo any processing to induce the enzyme synthesis. Hence, this is probably due to the time gap of adding the ONPG into the four tubes. The addition of ONPG should be performed continuously and immediately to prevent the time gap that may influence the result. There are several precaution steps to be noted in this experiment. First, the time gap for adding the reagents to the reaction tubes must be minimized as much as possible to reduce the occurrence of error in results. Furthermore, the sodium deoxycholate and toluene are toxic, so the addition of them must be performed under fume hood.

Conclusion: In a nut shell, IPTG is a better inducer for E.coli than lactose because IPTG can remain its concentration as it is not degraded by cells and it does not need any processing for induction. Lactose needs to be converted to allolactose first before inducing the production of -galactosidase, lactose permease, and lactose transacetylase. The results that obtained are not desirable and fit to the theory as the time gap during adding of ONPG into different tubes.

References: 1) 2) 3) 4) 5) 6) http://www.encyclo.co.uk/define/Allolactose http://www.mun.ca/biochem/courses/3107/Topics/Lac_positive_control.html http://www.ncbi.nlm.nih.gov/pmc/articles/PMC220091/ http://webbook.nist.gov/cgi/cbook.cgi?ID=C369073&Units=SI http://www.accessexcellence.org/RC/VL/GG/induction.php Lab Manual