Demonstrating lacZ Gene Regulation and Expression in E.Coli Under Various Conditions

Demonstrating lacZ Gene Regulation and Expression in E.Coli Under Various Conditions

Introduction

Numerous studies have been completed on gene expression of β-Galactosidase by utilizing sensitive lacZassays. Researchers were able in one study to detect which genes showed progressive hormonally regulated tumor promoter function in mammalian cells (Hall et al,1983). By doing so, this has further progressed research done on β-Galactosidase. To say what remains unknown about β-Galactosidase is hard to define, studies have already been done on cloning regulatory genes, discriminating transcriptional from posttranscriptional control and cloning target genes successfully (Silhavy et. al, 1985). Using these studies as fundamental information, scientists can use these results to supplement research into utilizing the lacZ enzyme for further studies.

Testing the effects of gene function based on how promoter elements control different aspects of gene expression in the Lac Operon is something that is greatly studied (Dickson, Abelson, et al 1975). The Lac Operon functions by using genes and regulatory elements called the Promoter, Operator, Repressor, β-Galactosidase and Permease to break down lactose for energy. The Promotor is the binding site of the Lac Operon; the Repressor is able to bind to the Operator to turn the operon off; β-Galactosidase cleaves lactose and Permease transports lactose into the cell. By cleaving lactose, which is a disaccharide, it is cut into either allolactose or galactose. Using positive and negative regulation, the Lac Operon can turn on and turn off depending on which sugars are present (Dickson, Abelson et al ,1975). Glucose is the preferred sugar for energy because it does not take much to break down, therefore repressing the lac operon when present, lactose, on the other hand is cleaved into galactose which takes more energy to break down. The Lac Operon is inducible by lactose or its analogs. (Dickson, Abelson et al, 1975).

By regulating gene expression in the operon, this allows cells to not waste energy metabolism on lactose if glucose is present. Positive regulation, also called Catabolite Repression, uses the cAMP-CAP protein complex to activate transcription. Lactose or analogs bind to the repressor and dissociate from the operator, having transcription to turn on, whereas negative regulation allows the repressor to bind to the operator to stop any transcription from occurring (Dickson, Abelson et al, 1975). When glucose is high, there would be no cAMP present, versus having low amounts of glucose which allows for cAMP to be present. Using an enzyme called beta-galactosidase, gene activation and function is measured with the substrate ONPG which turns yellow from a colorless solution after the β-Galactosidase enzyme is present. The lacZ protein, which is the structural gene for beta-galactosidase, catalyzes the reaction of cleavage of ONPG and yellow color change. Adding IPTG, which is an analog of lactose, induces gene expression. The environment best used for seeing this would be E. Coli, which is a great bacterium to use in experiments because of easy genetic manipulation and studying.

In the four experiments conducted, the hypotheses for each are: that lacZ⁺ activity would differ between the IPTG plate and control plate, showing more activity with IPTG in experiment one; that lacZ^– activity will be expressively lower compared to lacZ⁺ activity in experiment two; that increased IPTG will have more β-Galactosidase activity with glucose because there would be no regulation and no enzyme; and that lacI^S would have no color at all in either plate while lacO^C would have the strongest blue colors on both plates.

Materials and Methods

Experiment One

In total, four experiments were run for the β-Galactosidase assay. As an introductory experiment, 1 ml samples from mid log plate cultures of lacZ+ control and lacZ+ with 1mM IPTG were split into two test tubes each (so four tubes total), the control was labeled A and IPTG tubes labeled B. Tubes A received 0.2 ml of water and the IPTG B tubes received 0.2 ml of 0.013M ONPG. After a 37°C water bath for 20 minutes, 2.70 ml of 1M Na₂CO₃ was added to all tubes to stop the reaction. 1 ml of each tube was transferred to the cuvettes and A cuvettes were used to blank the spectrophotometer which was set at 420 nm. The B cuvettes were read to quantify the β-Galactosidase activity.

Experiment Two

The second experiment follows the same protocol as experiment one, the only difference this time is lacZ- and lacZ+ cultures were used. Every 30 minutes after inducing with 1mM IPTG, a new 2 ml sample was taken until 90 minutes was reached and underwent the same steps as experiment one to measure β-Galactosidase activity over time.

Experiment Three

Experiment three tested how much IPTG is necessary for full expression or what effect glucose has. LacZ+ cultures were used and split in 6 flasks. Flask 1 had no additions, flask 2 had 1 µM IPTG stock added, flask 3 had 10 µM IPTG stock, flask 4 had 100 µM IPTG stock, flask 5 had 10 mM glucose stock and flask 6 had 100 µM IPTG stock and 10 mM glucose stock added. Calculations of mmol ONP and enzymatic activity units were calculated by 420÷ 0.004= # mmol and then by taking that #mmol and ÷ by 20 to get the activity. After 60 minutes of incubation in 37°C, 2 ml samples were put in cuvettes and read at 420 nm in the spectrophotometer.

Experiment Four

Finally experiment four used 4 strains: CA800 (lacZ+), 30SO (lacZ-), CA7089 (lacO^c), and YA694 (LacI^S). Two plates were used, LB/Xgal and LB/Xgal+IPTG plates. Each plate was divided into four sections and a sterile toothpick was used to streak all four strains onto different sections of both plates. Both plates were inverted and put into the 37°C incubator to be retrieved 18 hours later. The strains were not labeled intentionally so that the plate color results would allow researcher prediction of the lac strain genotypes.

 Results and Discussion

 In experiment one, responses of β-Galactose activity to different liquid cultures was measured. By using lacZ⁺ control and lacZ⁺ IPTG as the substrates, β-Galactosidase showed a selective difference in amount of activity in the control culture versus the IPTG culture. IPTG can induce expression of the operon by acting like lactose, which then increased activity levels of β-Galactose in that culture. The hypothesis for this experiment was that lacZ⁺ activity would differ between the IPTG plate and control plate, showing more activity with IPTG. LacZ⁺ with IPTG showed larger levels of activity compared to lacZ⁺ control, due to this functional wild type gene being able to utilize IPTG to induce the lac operon activity. LacZ⁺ in IPTG exhibited 7.275 units of activity compared to only preforming 0.6875 units in the control which does support the initial hypothesis. This shows the significant difference and selective importance of substrates to be used to successfully induce β-Galactosidase activity. By carrying out this initial experiment, fundamental understanding of how lacZ⁺ preforms as a wildtype functioning gene primes understanding for these further experiments that test how different mutants of lacZ respond to different conditions.

Experiment 2 continues this idea of inducing β-Galactosidase activity by taking cultures that have been growing for 30-minute intervals and measuring much β-Galactosidase is produced. Hypothesizing that lacZ^– activity will be expressively lower compared to lacZ⁺ activity was the initial prediction made before conducting this experiment. Interestingly enough, the lacZ^– expression was very minimal to nonexistent compared to the lacZ⁺ activity, which supported this hypothesis. Figure 1 shows the influence of IPTG on the activity levels between the two cultures, highlighting the large difference of lacZ⁺ and lacZ^– activity over time.  lacZ^– showed 0 units at 90 minutes while lacZ⁺ showed 1.2125 units at 90 minutes. This could be due to the lacZ^– mutant gene being unable to make the β-Galactosidase enzyme successfully, whereas lacZ⁺ is a wildtype functional gene that is successfully induced thus making the β-Galactosidase enzyme.

 Experiment three measures β-Galactosidase activity in different cultures such as glucose and IPTG. Using glucose, which is the preferred sugar for E. coli versus lactose, really benefits understanding of how the promoter is positively and negatively regulated in the lac operon. It was hypothesized that increased IPTG will have more β-Galactosidase activity with glucose because there would be no regulation and no enzyme. Figure 2 shows the influence that the different substrates have on activity, displaying a trend of decreased activity in presence of glucose and increased activity with 100µM IPTG present which supports the hypothesis made before. In sample 4, activity was at 0.725 units compared to glucose which stayed at a low 0.125 in sample 5. This is due to the lac operon lacking positive regulation by the presence of glucose. The operon shuts down due to the repressor binding to the operator in the absence of cAMP/CAP. Whereas IPTG, which acts like lactose, depresses the lac operon by preventing repressor binding to Operator to break down the lactose for energy by using CAP/cAMP. This makes sense as a trend to have activity minimal in the presence of glucose. Similar results were found in another study, that IPTG shows higher levels of activity with increased levels of IPTG, similarly to the data presented earlier and in Figure 2 (Fernández-Castané et al, 2012).

Experiment four takes three unknown mutants of lacZ and wildtype lacZ to be grown on two E. coli plates, which are included in Figure 3. The hypothesis for this was that lacI^S would have no color at all in either plate while lacO^C would have the strongest blue colors on both plates. Table 1 takes the observed color intensities and assigns genotypes as well as further explanation supporting these genotyping claims below. In Table 1, it was observed that lacI^S actually did show faint blue color in the Xgal plate and no color in the IPTG X gal plate, which Dr. Spingola said to expect. This could be due to having a functional lacZ gene that could still make a small amount of β-Galactosidase despite being repressed. The lacO^C however showed the bluest intensity on both plates, due to constitutive expression, thus partially supporting the hypothesis. LacZ^– had no blue shown at all, this could be due to a frameshift mutation, whereas lacZ⁺ showed blue in both plates, but more blue in the IPTG plate. One confounding variable could be whether there was any lactose in the LB plates or cross contaminating samples.

These four experiments built off each other to increase understanding of how β-Galactosidase activity changes with different substrates, amount of time between cultures and concentration of IPTG. By learning more about how lacZ activity is influenced, especially with negative and positive regulation, additional studies can be conducted to grasp the diversity of this concept. Some studies that could be taken further could be testing different types of bacteria to see the response levels with and without IPTG present with different temperature levels. Measuring the effects of temperature on initiation of transcription would be very interesting for future studies.

Figures and Tables

Figure 1: β-Galactosidase activity over time is summarized in this figure. X-axis is the time points in minutes and Y-axis is the level of activity or expression of lacZ^– and lacZ⁺ in units.

Figure 2:β-Galactosidase activity in different concentrations of IPTG, Glucose or IPTG and Glucose combined is shown in this figure. X-axis shows concentration of plates in µM and mM and Y-axis shows activity of β-Galactosidase in units.

Table 1: Shows IPTG and non IPTG Xgal plate data for strains 1-4. This is depicted by -, +, ++ and +++ signs. – is given when no color is observed. + is given when a small concentration of blue is observed. ++ is given when a medium concentration of blue is observed. +++ is given when a large concentration of blue is observed. Below this is the four genotypes of the strains that are surmised from these observations as well as the reasoning as to why it is believed that strain is that genotype.

Figure 3: These are the Xgal plates with either IPTG present or without IPTG. The plates were divided into four quadrants for each individual strain to be smeared on each plate for observation.

Literature Cited

• Alfred Fernández-Castané, Glòria Caminal and Josep López-SantínEmail. Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures. Microb Cell Fact. 2012 May 9;11:58. Retrieved from https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-11-58#Sec
• Hall, C. V., Jacob, P. E., Ringold, G. M., & Lee, F. Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet. 1983;2(1):101-9. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/6302193
• Robert C. Dickson, John Abelson, Wayne M. Barnes and William S. Reznikoff. Genetic Regulation: The Lac Control Region. Science. 1975 Jan 10;187(4171):27-35. Retrieved from: https://www.jstor.org/stable/pdf/1739020.pdf?refreqid=excelsior%3A27812a31a78a1d391449ce9d69b93f6a
• Silhavy, T. J., & Beckwith, J. R. Uses of Lac Fusions for study of biological problems. Microbiol Rev. 1985 Dec;49(4):398-418. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC373045/pdf/microrev00059-0046.pdf