Western Blot Analysis to Illustrate Relative Control Levels of the lac and ara Promoters in Escherichia coli

Transcription

1 2007 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 35, No. 2, pp , 2007 Laboratory Exercises Western Blot Analysis to Illustrate Relative Control Levels of the lac and ara Promoters in Escherichia coli Received for publication, August 18, 2006, and in revised form, September 25, 2006 Brent L. Nielsen, Van C. Willis, and Chin-Yo Lin From the Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah The lactose operon and its control is a fundamental transcriptional regulatory concept presented in introductory and many advanced molecular biology courses. Much is known about the positive and negative control mechanisms that govern levels of expression of this operon. One basic principle that is taught about the lac operon is that it is leaky, meaning that the transcriptional control of the operon is not 100% efficient and that in wild-type cells, transcription from the promoter is never completely off, but there is always some basal transcription. In contrast, the arabinose operon is often used as an example of a tightly controlled operon, and transcription from the ara promoter is very low in the absence of inducer. The relative levels of control of these two operons can be illustrated using Western blots of proteins expressed in the presence and absence of the appropriate inducers and antibodies against the gene products. Different times of growth and the addition of inducer can also be examined. The results are very dramatic and help to reinforce the principles of promoter control. Keywords: lac operon, ara operon, Western blots, transcriptional control. The lactose (lac) operon was the first to be discovered and characterized and has become a paradigm for genetic control of transcription in bacteria [1]. This operon includes three genes; lacz encodes -galactosidase, lacy encodes lactose permease, and laca encodes a transacetylase (Fig. 1A). The lac operon is controlled by both positive and negative regulation involving CAP-cAMP 1 and the Lac repressor, respectively [2, 3]. The repressor is encoded by the laci gene under control of its own promoter. In the presence of glucose and/or the absence of lactose in wild-type Escherichia coli cells, the operon is down-regulated and considered to be off. The operon is activated in the presence of lactose or synthetic lactose analogs under conditions where glucose levels are low, which activates CAP-cAMP [3]. Thus, high levels of transcription of the lac operon occur only under the appropriate conditions of lactose availability and the absence of glucose (Fig. 1). However, it has been known from very early studies that the control of the lac operon is not completely efficient and exhibits leaky expression even in the presence of glucose and absence of inducer [2]. This is termed basal expression and is required to allow some lactose permease to be made so that it can import lactose into the cell when this sugar is available. It was originally thought that there was a single operator site for Lac repressor binding, but subsequently, it was determined that there are actually To whom correspondence should be addressed. Tel.: ; Fax: ; brent_nielsen@byu.edu. 1 The abbreviations used are: CAP, catabolite activator protein; GFP, green fluorescent protein; IPTG, isopropyl-1-thio- -D-galactopyranoside; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HRP, horseradish peroxidase; IP, immunoprecipitation. three operator sites in this operon [3]. In addition to the operator overlapping the promoter, there is an operator within the lacz gene and another upstream of the lac operon promoter, near the laci promoter [3]. These concepts are integral in introductory molecular biology and other similar courses [1], and students are exposed early to this operon when studying gene expression in bacteria. Leaky gene expression occurs due to a combination of the following reasons [4]. First, the Lac repressor protein does not bind to operator sites in the DNA with 100% efficiency, and thus, cannot completely shut off gene transcription from the lac operon promoter. The lac promoter, due to its high degree of characterization, is often used in plasmid cloning vectors for expression of recombinant proteins in bacteria. This promoter provides the ability to allow high levels of expression of an introduced gene. For expression cloning, constructs are usually made in a midto-high copy number plasmid (such as the puc, pbluescript, and pet vectors and their derivatives) to increase copy numbers of the recombinant gene. However, this has the potential to increase the leaky expression due to the high copy number of the gene, promoter, and operator sites. In addition, in plasmid constructs, there is the possibility of readthrough transcription of the inserted gene from other promoters on the plasmid [4]. The leaky expression appears to increase substantially as the cells enter stationary phase when grown in a complex medium (such as LB, commonly used for growth of E. coli host strains) and may be a response to nutrient limitation by the cells [5]. The arabinose (ara) operon is also often discussed in molecular biology courses and shows similarities and dif- This paper is available on line at DOI /bmb.25

2 134 BAMBED, Vol. 35, No. 2, pp , 2007 FIG. 2. The ara operon. A, repressed state. B, induced state. Based on Weaver and others [1, 6]. pol, RNA polymerase. FIG. 1. The lac operon. A, repressed state. B, induced state. Based on Weaver and others [1 3]. pol, RNA polymerase. ferences when compared with the lac operon [6]. Like the lac operon, the ara operon is regulated by glucose levels through the CAP-cAMP complex. However, the AraC regulatory protein, the product of the arac gene, exhibits both positive and negative regulatory activity (Fig. 2). The AraC protein in the absence of arabinose binds to specific operators to block RNA polymerase binding and prevent transcription from the P BAD promoter. The distances between the control elements are much greater in this operon when compared with the lac operon, and it has been shown that DNA looping occurs after binding of regulatory proteins to bring the elements into juxtaposition to provide the needed interactions [6]. The conformation of the AraC protein changes dramatically in the presence or absence of arabinose. In the presence of arabinose, the AraC protein forms a dimer to bind to inducer sites in the operon to facilitate RNA polymerase binding (Fig. 2). In the absence of arabinose, AraC also functions as a dimer (but with a different conformation) and binds to an upstream operator to cause DNA looping and block RNA polymerase binding [1, 6]. As a result, this promoter has been shown to exhibit a much tighter degree of control when compared with the lac promoter, even when the ara promoter is placed in a high copy number plasmid. This promoter is often regarded as having all or nothing expression [7] and is included in many bacterial expression vectors when the gene product is thought or known to be toxic to the bacterial host strain. In this case, the host strain containing the gene construct can be grown up to high density before adding the arabinose inducer, at which time the recombinant protein begins to be expressed. However, it has been shown that for high copy number plasmids in a strain grown to high cell density, arabinose levels may not be sufficient to fully induce all copies of the DNA. This could result in a mixed population of fully induced and completely uninduced cells [8]. In this case, some expression may occur, but it may be low depending on the proportion of uninduced cells in the population. A mutant strain defective in arabinose uptake and degradation has been utilized to develop an expression system that shows more than 100-fold modulation of gene expression from the P BAD promoter in a vector with a tightly controlled copy number [9]. We have developed an exercise for a molecular biology laboratory methods course that illustrates these basic concepts of transcriptional regulation from the lac and ara operons. Using appropriate strains, plasmids, and antibodies, students conduct Western blots to examine the levels of basal and induced expression of proteins from the lac and ara promoters. The results allow a clear observation of the relative amounts of uninduced versus induced levels of expression from each of the promoters. Immunoprecipitation was included for analysis of the arabinose operon to detect small amounts of protein in the absence of inducer. MATERIALS AND METHODS Vectors and Host Strains E. coli DH5 and HB101 strains were used for analysis of lac and ara promoters, respectively. The DH5 strain has a chromosomal deletion of the lac operon and the lacz M15 portion of the lac operon included in the chromosome as part of an integrated phage. This insertion encodes most

3 135 of the -galactosidase gene (termed the -peptide) and the entire permease gene and is missing just a small portion of the 5 end of the -galactosidase gene (termed the -peptide). This results in a protein product that is slightly smaller than the full -galactosidase but is still recognized by a polyclonal antibody against -galactosidase. This portion of the lac operon is thus present as a single copy on the chromosome and not part of a plasmid that may have a much higher copy number relative to the chromosome. This construct contains the wild-type operator and promoter for the lac operon and exhibits the usual level of control by glucose and inducer. For analysis of ara promoter control, the pglo plasmid from Bio-Rad was used in the HB101 E. coli host strain. In the pglo plasmid, a gene encoding GFP from Aequorea victoria is placed under control of the ara P BAD promoter. For this construct, induction occurs with the addition of arabinose to the medium, which causes the cells to fluoresce green under ultraviolet light. For both strains, cultures are grown in LB broth, which is a rich medium that lacks glucose, at 37 C with shaking. Two ml of LB broth is inoculated with a single isolated colony from a freshly streaked culture and grown either overnight (to stationary phase) or for a set time as designated in the figure legends. Induction of Strains and Isolation of Total Proteins Each strain is grown as described above in the presence or absence of inducer. For the DH5 strain, IPTG (Sigma) is added to a final concentration of 1 mm. For HB101/pGLO, arabinose was added to a concentration of 6 mg/ml. After the desired time of growth, 1.5 ml of culture is transferred to a microcentrifuge tube, and the bacteria pelleted by centrifugation at maximum speed for 1 min. The cells are then resuspended and lysed in 100 l of1 SDS- PAGE buffer [10] followed by heating at 95 C and centrifuging for 5 min to pellet debris. The supernatant is transferred to a clean tube, and 10 l of each sample is loaded into adjacent lanes of a 10 20% precast polyacrylamide gel (Bio-Rad). Proteins are separated by electrophoresis in Tris-glycine-SDS buffer [10] for 45 min at 150 V. Protein molecular weight markers (Bio-Rad) are included in the gel and may include prestained or labeled proteins. Gloves should be worn at all times when handling the gels and membranes. Immunoprecipitation 50 l of1 SDS-PAGE buffer [10] is added to each bacterial pellet. The sample is pipetted up and down until the pellet is dissolved and then centrifuged at 13,000 rpm at 4 C in a microcentrifuge for 30 min to clarify the lysate. The clarified lysate (supernatant) is transferred to a new 1.5-ml tube and suspended in 1 ml of immunoprecipitation (IP) buffer (PBS, ph 7.4, 1% Triton X-100), 5 l of rabbit anti-gfp antibody (Invitrogen), and 50 l of protein A/G agarose beads (Santa Cruz Biotechnology) and incubated for 90 min at 4 C on a rotator in the cold room. Following incubation, beads are washed three times with 1 ml of IP buffer. Washed beads containing the immunocomplex are then resuspended in 50 l of SDS-PAGE lysis/ loading buffer. Transfer of Proteins to PVDF Membrane While the gels are running, a piece of PVDF membrane cut to the same size as the gel is briefly soaked in methanol followed by soaking for at least 20 min in anode buffer (12 mm Tris, ph 9.6, 8 mm CAPS, 15% v/v methanol). After electrophoresis, gels are soaked in cathode buffer (12 mm Tris, ph 9.6, 8 mm CAPS, 2% SDS) for min. A transfer sandwich is then assembled from bottom to top as follows: one sheet of thick blotting paper soaked in anode buffer, the PVDF membrane, the gel, and at the top, a sheet of thick blotting paper soaked in cathode buffer. A Pasteur pipette can be rolled across the surface of the assembly to remove air bubbles. The transfer sandwich is then placed on the bottom plate of a semi-dry transfer blotter (Bio-Rad), and the stainless steel cathode cover is placed on the assembly and locked in place. Transfer of proteins from the gels to the membranes is performed at 1.5 ma/cm 2 of gel for min. Efficiency of transfer can be observed by the amount of prestained protein markers transferred or by Coomassie Blue staining the gel after disassembly of the transfer sandwich. Transfer can also be performed in a tank TABLE 1 blotting apparatus [10]. Western Blot Analysis Membranes are blocked in 5% nonfat dry milk in TBS (50 mm Tris, ph 7.4, 200 mm NaCl) for 1hatroom temperature to prevent nonspecific binding to antibody. Antisera against -galactosidase (from mouse, Sigma) or against GFP (from rabbit, Invitrogen) is diluted 1:2000 in blocking buffer and added to the membrane in an appropriate container (we have found that a pipette tip box lid works very well with 25 ml of solution). The membrane is incubated with the diluted antisera overnight with gentle agitation at 4 C. Each membrane is then washed three times for 15 min each with TBS, with the second wash also containing 0.1% Tween 20 to reduce background. The membranes are then blocked as before (this blocking time can be reduced to 20 min). Development of Western Blots The Pierce SuperSignal West Pico chemiluminescent system was used to detect the HRPlabeled secondary antibody bound to the primary antibodies on the membrane. Development was carried out according to the directions from the manufacturer (Pierce). Membranes are incubated with the secondary antibody (1:20,000 dilution of antimouse- or anti-rabbit-horseradish peroxidase HRP (Promega) in blocking solution) for 1 h at room temperature followed by washing three times with TBS as above (second wash also contains 0.1% Tween 20). Immediately before use, equal volumes of the stable peroxide solution and the luminal/enhancer solution are mixed. Typically, about 6 ml of solution total is used for each 7 8-cm membrane, and the solution is spread on the surface of a piece of plastic wrap on the bench top to minimize the volume required. The membrane is placed protein side down on the solution for 5 min at room temperature. The membrane is then lifted using forceps, and excess liquid is drained away or blotted with tissue paper and wrapped in a clean piece of plastic wrap. The wrapped membrane is exposed to x-ray film in the darkroom for 2 30 s and developed. Handling and Disposal of Materials Care should be taken when handling the polyacrylamide gels to prevent tearing of the gel material. Gloves and lab coats should always be worn when working with the samples, gels, and membranes. If gels are stained with Coomassie Blue or another dye, the spent dye and destain solutions should be disposed of according to local requirements. Many of these solutions are acidic and should be disposed of according to the campus chemical safety guidelines. Other solutions are near neutral ph, and in some locations, can be disposed of in the laboratory sink (be sure this is permissible in your locations before doing so). Precast polyacrylamide gels do not pose any significant hazard, but if gels are prepared locally, then great care should be taken in handling the polyacrylamide reagents, especially if they are in powder form, as these chemicals are neurotoxins. Guidelines for handling can be found in most laboratory manuals [10].

4 136 BAMBED, Vol. 35, No. 2, pp , 2007 FIG. 4.Western blot of -galactosidase expression in overnight (stationary phase) cultures. As can be seen, there appears to be less difference between the uninduced and induced protein samples. FIG. 3.Western blot analysis of -galactosidase expression in induced and uninduced bacterial cells. A, DH5 cells were incubated in the presence ( ) or absence ( ) of IPTG inducer as described under Materials and Methods for the times indicated at the bottom of the figure. Total protein was recovered and separated by SDS-PAGE, transferred to PVDF membrane, and incubated with mouse anti- -galactosidase antibody. Detection was performed by chemiluminescence using an anti-mouse-hrp antibody. B, Coomassie Blue-stained gel showing total protein from induced ( ) and uninduced ( ) cells. The gel was loaded identically as the gel used for the Western blot shown in A. M, marker proteins included in the gel (Bio-Rad Precision unstained markers, with molecular weights from top to bottom of 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kda). RESULTS AND DISCUSSION This laboratory exercise takes 2 or 3 laboratory periods to complete (see Table I for time line). On the first day, liquid LB broth cultures (2 ml) are inoculated from isolated colonies on plates previously streaked out and incubated by the teaching assistant or instructor. At the desired times (either along with the initial inoculation, which is what we have routinely done, or after a specific time of growth), inducer is added to the culture (IPTG for the lac operon in DH5 cells, arabinose for the pglo plasmid in HB101 cells), and incubation is continued at 37 C with shaking. After the designated time of incubation (5 or 6 h to overnight), 1.5 ml of culture is transferred to a microcentrifuge tube, and the cells are pelleted by centrifugation at maximum speed for 2 min. Upon resuspension in 1 SDS loading buffer, the samples can be directly loaded into polyacrylamide gels for electrophoresis or stored at 20 C until use. Electrophoresis and processing can be carried out on the same day, but if so, the transfer of proteins to the PVDF membrane needs to also be done the same day. After transfer, the membrane can be stored at 4 C for 1 or 2 days, or it can be taken directly to the blocking step followed by overnight incubation with the primary antibody. These steps can all be carried out in one period of about 3 h or divided into 2 days. The washing, FIG. 5. Western blot analysis of GFP expression from an arabinose-inducible promoter. The HB101 host strain carrying the pglo plasmid was grown in the presence ( ) or absence ( ) of arabinose inducer overnight, total proteins were isolated as before (for the left panel, labeled L for lysate), and samples were separated by SDS-PAGE. Proteins were then transferred to a PVDF membrane and incubated with rabbit anti-gfp antibody as described under Materials and Methods. The right panel includes samples grown in the presence ( ) or absence ( ) of inducer overnight or for 5 h, as indicated, after concentration by IP. M is a marker lane containing labeled proteins (the 30- and 40-kDa markers can be observed). The arrows indicate the 29- kda GFP protein size. The higher molecular weight bands are apparently due to a cross-reacting artifact from the cells. incubation with secondary antibody, and development using the chemiluminescent substrate should all be done in 1 day and should also take about 3 h. Typical student results for analysis of lac operon control are shown in Fig. 3A. As can be easily seen, there is an increase in signal for the -galactosidase protein in the presence of the IPTG inducer, but there is still a significant amount of signal in the absence of inducer. This difference is more noticeable for the samples grown for shorter times (5 or 6 h when compared with 10 h). This difference is not due to differences in total protein, as can be seen by comparison of the amount of protein in each lane in an identical, Coomassie Blue-stained gel (Fig. 3B). For overnight cultures, there is less difference in signal for -galactosidase between the uninduced and induced protein samples (Fig. 4). These results are in agreement with the published findings that control is decreased in stationary phase cultures [5]. Results from student Western blots of the arabinose operon are shown in Fig. 5. The first two lanes contain total proteins from cell lysates of overnight cultures grown in the presence ( ) and absence ( ) of arabinose, the inducer for the operon. The blot has been incubated with polyclonal antibodies against GFP, which is the product of the reporter gene linked with the arabinose promoter in the construct. A strong signal for the size of GFP is observed for the induced sample, whereas no detectable signal is observed in the uninduced lane. The higher molecular

5 137 weight band is an unrelated protein that cross-reacts with the antibody. Some samples were subjected to immunoprecipitation to concentrate the GFP, as can be observed by the very concentrated GFP band in the IP lane (Fig. 5). For the overnight culture grown in the absence of arabinose inducer, a band of the appropriate size is detected, but it is much less intense when compared with the induced sample lane. The band is nearly undetectable in the uninduced sample grown for only 5 h. Variations are observed between samples and may be a result of differences in the length of time of growth until and after induction and small differences in the temperature of incubation. Other post-incubation differences could also lead to variations, including insufficient pelleting or resuspension of the bacterial cells, incomplete denaturation of proteins prior to SDS-PAGE, errors in gel loading, and transfer or post-transfer steps. Potential Problems/Troubleshooting 1. Weak signals may be due to one or more of the following: insufficient transfer from gel to membrane, incorrect transfer buffers, incubation with antibody was not long enough, or antibody is too dilute. 2. Too much background can result from using too much antibody or from allowing the membrane to dry out during the incubation steps. Enough solution should be used to keep the membrane completely submersed but without wasting excess solution and antibody. We have found that a pipette tip box lid is an ideal size for holding ml of solution. The proper dilution of antibody needs to be empirically determined. 3. Suboptimal immunoprecipitation conditions may lead to reduced protein signal or an increase in nonspecific bands and background staining following Western analysis. Salt and detergent concentrations in the IP buffer can be adjusted as needed to maximize efficiency and specificity. Questions for Classroom Discussion 1. What is the basis of any differences in the amount of -galactosidase observed in Western blots relative to time of growth? 2. Does there appear to be more control of gene expression in the logarithmic stage of growth versus stationary phase? 3. Why should the time when inducer is added to the culture affect expression levels if the cells are grown to the same final density? 4. How might temperature of culture incubation affect expression; what would be some possible reasons for altering the temperature? 5. Upon comparison, does the ara promoter show greater or lesser control over gene expression when compared with the lac promoter? 6. How does immunoprecipitation enhance detection levels of proteins? 7. When would lac promoter constructs have the advantage over ara promoter constructs? 8. For what applications would the ara promoter be most useful? 9. What modifications to either promoter might be useful to obtain optimal expression under specific growth conditions? SUMMARY The use of the lac and ara promoters in common host strain and vector constructs provides an illustrative exercise for comparison of the relative strength of control over gene expression from these two promoters. The results clearly show that the ara promoter exhibits considerably more control over gene expression when compared with the lac promoter. The relative differences in gene expression between logarithmic and stationary phases of bacterial growth are also observed. This type of exercise can be coupled with other techniques to teach additional concepts, such as site-directed mutagenesis of promoter sequences to alter gene expression, fusion of the promoters with other reporter genes or desired products, and purification. Acknowledgments We thank the molecular biology laboratory students over the years who have tested and helped refine these experiments. We also thank the Department of Microbiology and Molecular Biology and the College of Biology and Agriculture at Brigham Young University for financial support for supplies and equipment for this course. REFERENCES [1] R. F. Weaver (2005) Molecular Biology, 3rd Ed., McGraw Hill Inc., New York, pp [2] F. Jacob, J. Monod (1961) Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3, [3] W.S. Reznikoff (1992) The lactose operon-controlling elements: A complex paradigm, Mol. Microbiol. 6, [4] L. C. Anthony, H. Suzuki, M. Filutowicz (2004) Tightly regulated vectors for the cloning and expression of toxic genes, J. Microbiol. Meth. 58, [5] T. H. Grossman, E. S. Kawasaki, S. R. Punreddy, M. S. Osburne (1998) Spontaneous camp-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability, Gene (Amst.) 16, [6] R. Schleif (2000) Regulation of the L-arabinose operon of Escherichia coli, Trends Genet. 16, [7] R. M. Morgan-Kiss, C. Wadler, J. E. Cronan, Jr. (2002) Long-term and homogeneous regulation of the Escherichia coli arabad promoter by use of a lactose transporter of relaxed specificity, Proc. Natl. Acad. Sci. U. S. A. 99, [8] D. A. Siegele, J. C. Hu (1997) Gene expression from plasmids containing the arabad promoter at subsaturating inducer concentrations represents mixed populations, Proc. Natl. Acad. Sci. U. S. A. 94, [9] L. M. Bowers, K. LaPoint, L. Anthony, A. Pluciennik, M. Filutowicz (2004) Bacterial expression system with tightly regulated gene expression and plasmid copy number, Gene (Amst.) 340, [10] J. Sambrook, D. W. Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.