2nd Rotation Report
- 1. ΠΑΝΕΠΙΣΤΗΜΙΟ ΚΡΗΤΗΣ ΣΧΟΛΗ ΘΕΤΙΚΩΝ ΕΠΙΣΤΗΜΩΝ ΤΜΗΜΑ ΒΙΟΛΟΓΙΑΣ 2Η ΕΡΓΑΣΤΗΡΙΑΚΗ ΆΣΚΗΣΗ (ROTATION) ΕΡΓΑΣΤΗΡΙΟ ΚΡΥΣΤΑΛΛΟΓΡΑΦΙΑΣ Ετερόλογη έκφραση και χαρακτηρισμός της διοξυγενάσης c1 του τετράνυχου, Tetranychus urticae ΣΤΕΛΛΑ ΚΟΥΝΑΔΗ ΥΠΕΥΘΥΝΟΣ ΚΑΘΗΤΗΤΗΣ : Μ. ΚΟΚΚΙΝΙΔΗΣ ΗΡΑΚΛΕΙΟ ΟΚΤΩΒΡΙΟΣ 2015
- 2. 2 Abstract Tetranychus urticae is a polyphagous pest that causes problems in crops and its biological control is mainly based on the application of acaricides. However, the rapid development of resistance in acaricides is an important problem. Many studies have been focused on the elucidation of detoxification mechanisms on the molecular level. The c1 dioxygenase, which has been studied in this report, is one of the enzymes that were found to be differentially expressed in resistant strains of T. urticae. The study objective was the heterologous expression and molecular characterization of the c1 dioxygenase. An attempt was made to overexpress the c1 dioxygenase in E.coli cells and then to purify the recombinant protein, in order to proceed in further structural analysis of the molecule.
- 3. 3 Introduction Tetranychus urticae belongs to the family Tetranychidae (Arthropoda: Chelicerata: Arachnida: Acari) or spider mites. T. urticae is a polyphagous pest which causes problems in crops and leads to economical damages. The biological control of mites is a very important problem and is mainly based on the application of acaricides. However, the main consequence in mite control is the rapid development of resistance to acaricides. In order to analyze the detoxification mechanisms, many studies have been focused on resistance to acaricides in relation to detoxification genes, such as P450 monooxygenases, carboxyl/cholinesterases (CCEs), glutathione-S-transferases (GSTs) and dioxygenases (1, 2). In previous studies, resistant and susceptible, T. urticae strains were analyzed regarding the effects of acaricides on gene expression. Microarray experiments and qPCR analysis have indicated a set of differentially expressed genes. Among these detoxification genes are secreted protein molecules, identified as intradiol ring-cleavage dioxygenases (ID-RCDs) (3, 4). The c1 dioxygenase is one of these striking differentially expressed molecules and belongs to the subgroup of intradiol ring-cleavage dioxygenases. ID-RCDs catalyze the incorporation of the two atoms of molecular oxygen into catecholic substrates by cleaving the carbon-carbon bond between the two hydroxyl groups, allowing the degradation of aromatic rings. In this study, the objective was the heterologous expression and molecular characterization of the c1 dioxygenase of Tetranychus urticae. Specifically, the aim was the achievement of dioxygenase overexpression in E. coli cells, and afterwards, the protein purification using the His-tag epitope of the transgene and finally, the crystallization of the protein in order to define its structure.
- 4. 4 3. Materials and Methods 3.1 Expression plasmid construction The gene sequence (786bp) was synthesized from GenScript® (USA). The construction of the recombinant plasmid vector was performed through connection of the insert DNA into a compatibly digested pET26b+ vector. Ligation reaction has been previously conducted in the lab and the complete plasmid has been provided to us, in order to be transformed into bacterial cells for propagation. 3.2 Transformation of E.coli and selection of transformants E.coli BL21 DE3 competent cells were transformed with 10ng expression vector using the heat shock protocol. The transformant cells were plated on agar plates containing 100μg/ml of ampicillin. The plate was incubated at 37°C for 12-16 hours (overnight incubation). Several colonies appeared on the agar plate and 2 of them were selected for protein expression. 3.3 Plasmid preparation and Restriction enzyme digestion Plasmid DNA purification was performed using QIAprep Spin Miniprep Kit. 2 ml of liquid overnight E. coli culture in LB medium were used for plasmid purification according to the manufacturer’s instructions. Restriction enzyme digestion was performed in order to examine the purified plasmid. The restriction enzymes which used for digestion were NdeI and XhoI, from New England BioLabs. The digested and undigested controls were subjected to 1% gel electophoresis. 3.4 Expression of c1 dioxygenase Two different colonies were screened for containing the plasmid construction and the c1 clone was chosen for IPTG induction and protein expression. E. coli cells from c1 clone were grown in 5ml LB with 25μg/ml Kanamycin with vigorous shaking at 37°C for 12-16 hours (overnight incubation). The following day, 0.5ml of the overnight bacterial culture were inoculated in 9.5ml of LB medium containing 25μg/ml Kanamycin (1/20 dilution). The culture was incubated at 37°C for approximately 3 hours, until the absorbance at 600 nm (OD600) was between 0.4 and 0.6. When the correct OD has
- 5. 5 been reached, IPTG was added in a final concentration of 1mM. The culture was transferred at 28°C and incubated overnight (12-18 hours) with vigorous shaking. The following day, cells were harvested by centrifugation at 4000 rpm for10 min, RT. Pellets from induced and un-induced cells were resuspended in 50μl sample buffer and were analyzed using SDS-PAGE followed by Coomassie brilliant blue staining, as well as Western Blot analysis. 3.5 Purification of c1 dioxygenase E. coli cells from an overnight culture were inoculated in 2lt LB containing 25μg/ml Kanamycin. The culture was incubated at 37°C with vigorous shaking, until the OD at 600nm reached 0.4 -0.6. Then, the protein production was induced by adding IPTG to a final concentration of 1Mm, and the culture incubated at 37°C overnight. Bacterial pellet from an overnight IPTG-induced culture was resuspended in 40ml rinse solution (20mM Tris-HCl pH 8, 200Mm NaCl) and cells were pelleted again with centrifugation. The cell pellet was then resuspended in 50ml Lysis buffer (25mM Tris-HCl pH 8, 300Mm NaCl, 5mM Imidazole) with protease inhibitors (1mM PMSF, 150ng/ml benzamidine) and cells were disrupted with sonication (10 repetitions of 30 seconds sonication with intermediate incubation on ice). Cell suspension was centrifuged at 12500rpm, 4°C for 1 hour. The soluble fraction (supernatant) containing the dioxygenase c1 fused to the His6-epitope was loaded on a resin Ni2+ -NTA agarose column, previously equilibrated with Lysis buffer. The flow through was collected for SDS- PAGE analysis. The resin was washed with two solutions containing 10mM and 20mM imidazole, respectively, diluted in Lysis buffer, and each wash fraction was collected for SDS-PAGE analysis. Afterwards, the dioxygenase was eluted from resin in 10 fractions, with elution buffer containing 100mM and 500mM imidazole. Finally, the elution fractions were analyzed with Bradford and 12.5% SDS-PAGE stained with Coomassie blue, as well as Western Blot analysis.
- 6. 6 Results and Discussion At first, E. coli BL21 (DE3) cells were transformed with the plasmid vector that carries the optimized sequence of dioxygenase c1. The cells were plated on a LB-agar plate with ampicillin, as a selectable marker, and incubated at 37°C for 18 hours (overnight). The next day, the plate was full of colonies. Two of the colonies were selected and inoculated in 5ml of LB medium with kanamycin at a concentration of 25μg/ml. The cultures were incubated at 37°C for 18 hours (overnight). Afterwards, a plasmid preparation was performed using these bacterial cultures and then, the extracted plasmid DNA was digested with the restriction enzymes NdeI and XhoI, in order to check for the right insert DNA at 786 bp. The digestion samples were electrophorated in 1% agarose gel (Figure 1). Based on digestion results, it was decided to use the first clone in order to continue for the expression procedures. The DNA zones are not very sharp because the cells used for plasmid preparation were not suitable for giving large amounts of plasmid DNA. Furthermore, glycerol master stock were prepared for the two clones using the previous mentioned cultures. Subsequently, 4 flasks, each containing 100ml LB medium with 25μg/ml kanamycin were inoculated with 5ml bacterial pre-culture of clone 1 and were incubated at 37°C for approximately 3 hours, until the absorbance at 600 nm (OD600) was between 0.4 and 0.6. The protein production was induced by adding IPTG reagent to a final concentration of 1mM, and the culture was incubated at 16°C, 23°C, Figure 1. Comparison between digested pET26b+ plasmids from 2 colonies of BL21 (DE3) cells. The digestion was performed with the restriction enzymes NdeI and XhoI. The insert’s size is 786 bp.
- 7. 7 28°C and 37°C overnight. The following day, bacterial cells were pelleted with centrifugation, resuspended in Lysis buffer and disrupted with sonication. The pellet and soluble fractions were collected for SDS-PAGE analysis (Figure 2). Based on the results, no protein overexpression is detected neither at the four different temperatures nor on incubation time. Thus, the next step was the picking up of four new colonies from the agar plate with the transformed cells. The following expression experiment performed in small scale. Four new colonies were inoculated in 5ml LB with 25μg/ml kanamycin and incubated at 37°C overnight. In addition, a 5ml culture with cells from the previous master glycerol stock was also incubated. The next day, flasks with 15ml of LB with 25μg/ml kanamycin were inoculated with 1/20 volume of the bacterial pre-culture and incubated at 37°C until the OD600 was about 0.4 to 0.6. The protein production was induced by adding IPTG to a final concentration of 1mM, and the culture was incubated overnight at 37°C. Subsequently, crude samples of induced and un-induced cells were analyzed on 12.5% SDS-PAGE (Figure 3). Figure 2. Comparison between the pelleted and the soluble fraction of disrupted BL21 (DE3) cells after IPTG induction. Cells were incubated at 16°C, 23°C, 28°C and 37°C for 4 hours or overnight incubation.
- 8. 8 Similarly, no protein overexpression is detected between the inducted and un-inducted samples. Thus, a western blot analysis was performed with an Anti-His antibody, in order to detect the c1 dioxygenase fused to His-tag, and check if the protein is expressed even in small quantity. The previous samples were electrophorated in a 12.5% SDS-Polyacrylamide gel and then, transferred in a PVDF membrane. The membrane was covered with blocking solution (BSA in TBS) for 1 hour, and then incubated overnight with Anti-His antibody in 1/1200 dilution. The detection of protein was achieved after incubation of membrane with staining solution (NBT/BCIP solution) (Figure 4). A purified known protein (~97 kDa) which was provided by lab members used as control. Western blot worked well for the control protein, but not for c1 dioxygenase, which is not detected in none of the five clones. Because of that, it was decided to pick up more colonies from the transformation plate and induce the protein production with IPTG from a new aliquot. Figure 3. Comparison between the crude samples of induced and un-induced BL21(DE3) cells. After addition of IPTG, cells were incubated at 37°C overnight. Figure 4. Comparison between the crude samples of induced and un-induced BL21(DE3) cells. Protein detection was achieved with Anti-His antibody. After addition of IPTG, cells were incubated at 37°C overnight. For positive control, it was used a purified protein of known molecular weight.
- 9. 9 Ten new colonies were picked up and grown in small scale, as described previously. A new IPTG aliquot was used at a concentration of 1mM for induction of protein expression and the cultures was incubated overnight at 37°C. Subsequently, crude samples of induced and un-induced cells were analyzed on 12.5% SDS-PAGE (Figure 5). After the SDS-PAGE analysis, all samples that checked seemed to overexpress the c1 dioxygenase. The following step was to perform a western blot analysis in order to detect the dioxygenase with the Anti-His antibody. As described previously, crude samples were electrophorated in a 12.5% SDS-Polyacrylamide gel and then, transferred in a PVDF membrane. Then, membrane was incubated overnight with Anti-His antibody in 1/1200 dilution, and the detection of protein was achieved by soaking the membrane with staining solution (NBT/BCIP). A purified protein (~97 kDa) which was provided by lab members used as control. (Figure 6). Western blot data confirmed the overexpression of dioxygenase for all 7 colonies checked. As shown, all samples had a protein zone in western blot, in contrast to un-induced sample, which hadn’t been induced with IPTG. Clone 5 was selected for the following experiments because of the dense zone in western blot. Glycerol stocks were prepared for clone 5 and 6 and stored at -80°C. Figure 5. Comparison of protein expression among 10 bacterial BL21 (DE3) clones. Crude samples of induced cells compared with un-induced cells. Cells were incubated at 37°C overnight, after addition of IPTG.
- 10. 10 Clone 5 was selected because of its dense protein zone in western blot, so the next step was to screen different growth conditions for protein expression. This expression experiment performed in a larger scale. Cells of clone 5 from the glycerol stock were inoculated in 25ml LB with 25μg/ml kanamycin and incubated at 37°C overnight. The next day, three flasks, each containing 100ml LB with 25μg/ml kanamycin were inoculated with 1/20 volume of the bacterial pre-culture and incubated at 37°C until the OD600 was about 0.4 to 0.6. The protein production was induced by adding IPTG to a final concentration of 1mM, and then the 3 cultures were divided and incubated at different temperatures. The first culture was incubated at 20°C, the second at 28°C and the third at 37°C. Samples from the three cultures were collected 4 hours after the addition of IPTG. Cultures were incubated overnight. Subsequently, crude samples of induced (includes 4 hours and overnight incubation) and un-induced cells were analyzed on 12.5% SDS-PAGE (Figure 7). The results, surprisingly, didn’t show any overexpression of c1 dioxygenase, even if clone 5 had previously been tested with Anti-His antibody and found to express the enzyme. However, at the coomassie gel there is a zone at ~30kDa, which is clearly visible at 28°C and could be the zone of dioxygenase because the molecular weight is consistent with that of enzyme. A possible explanation is that the cells, indeed, express the c1 dioxygenase, but in low concentrations. Because of that, protein can be detected in Western Blot, which is a sensitive method, but not with coomassie stained gel, which shows, primarily, high concentration proteins and proteins which is overexpressed. Figure 6. Detection of c1 dioxygenase in crude samples of different induced BL21 (DE3) clones. Protein detection was achieved with Anti-His antibody. After addition of IPTG, cells were incubated at 37°C overnight. For positive control, it was used a purified protein of known molecular weight.
- 11. 11 In order to determine the concentration of the expressed c1 dioxygenase, it was decided to perform a similar experiment, but in large scale, with a bacterial culture of 2 litters, incubation at 28°C, which shown to be the optimal growth temperature and finally, purify the protein using Immobilized-metal affinity chromatography (IMAC), with a resin Ni2+ -NTA agarose column. Thus, a pre-culture of 100ml LB with 25μg/ml kanamycin was inoculated with cells of clone 5 from glycerol stock and incubated overnight at 37°C. The next day, 2 flasks, each containing 1L LB with 25μg/ml kanamycin were inoculated with 1/20 volume of the bacterial pre-culture and incubated at 37°C until the OD600 was about 0.4 to 0.6. IPTG was added in the cultures to a final concentration of 1mM, and then the 2 cultures were incubated at 28°C. The next day, cells were pelleted with centrifugation at 4500 rpm for 30 minute. Bacterial pellet was resuspended in 40ml rinse solution (20mM Tris-HCl pH 8, 200Mm NaCl) and cells were pelleted again with centrifugation. The cell pellet was then resuspended in 50ml Lysis buffer (25mM Tris-HCl pH 8, 300Mm NaCl, 5mM Imidazole) with protease inhibitors (1mM PMSF, 150ng/ml benzamidine) and cells were disrupted with sonication (10 repetitions of 30 seconds sonication with intermediate incubation on ice). Cell suspension was centrifuged at 12500rpm, 4°C for 1 hour. The soluble fraction (supernatant) containing the dioxygenase c1 fused to the His6-epitope was loaded on a resin Ni2+ -NTA agarose column, previously equilibrated with Lysis buffer. The flow through was collected for SDS-PAGE analysis. The resin was washed with two solutions containing 10mM and 20mM imidazole, respectively, diluted in Lysis buffer, and each wash fraction was collected for SDS-PAGE analysis. Afterwards, the dioxygenase was eluted from resin in 10 fractions, with elution buffer containing 100mM and 500mM imidazole. Figure 7. Comparison of protein expression at different temperatures. Clone 5 from the transformed BL21(DE3) cells induced with IPTG and incubated at 20, 28 and 37°C. Crude samples of induced and un- induced cells are compared.
- 12. 12 Finally, the elution fractions were analyzed with Bradford and 12.5% SDS-PAGE stained with Coomassie blue, as well as Western Blot analysis (Figure 8, Figure 9). The SDS-PAGE results indicate that the c1 dioxygenase is present in the soluble fractions, and especially in elutions 2 to 5. Subsequently, a western blot analysis with Anti-His antibody was performed in order to detect the dioxygenase and confirm the previous results. The Western Blot results confirmed the expression of c1 dioxygenase. Indeed, the protein is present in the soluble fractions and not in the pellet. However, the dioxygenase is not overexpressed, although Figure 8. Analysis of c1 dioxygenase purification with Immobilized-metal affinity chromatography (IMAC). The purification was performed with a resin Ni2+ -NTA agarose column. The washes were made with solutions containing 10mM and 20mM imidazole and the consecutive elutions with 100mM and 500mM imidazole. Figure 9. Analysis of c1 dioxygenase purification with Immobilized-metal affinity chromatography (IMAC). Protein detection was achieved with Anti-His antibody. For positive control, it was used a purified protein of known molecular weight, provided by the lab.
- 13. 13 the IPTG was added in a high concentration of 1mM. In this situation, this is a big problem because the aim of these experiments was to overexpress and purify the dioxygenase, in order to crystallize the molecule and perform further structural analysis of the protein. In order to overcome this problem, the following step was to transform a different E.coli strain, the C43 cells, and check if these cells are able to overexpress the dioxygenase. In order to do this, E. coli C43 cells were transformed with the plasmid vector that carries the optimized sequence of dioxygenase c1. The cells were plated on a LB-agar plate with ampicillin, as a selectable marker, and incubated at 37°C for 18 hours (overnight). The next day, the plate was full of colonies. Five colonies were selected and inoculated in 5ml of LB medium with kanamycin at a concentration of 25μg/ml. The cultures were incubated at 37°C for 18 hours (overnight). The next day, flasks with 10ml of LB with 25μg/ml kanamycin were inoculated with 1/20 volume of the bacterial pre-culture and incubated at 37°C until the OD600 was 0.4 to 0.6. The protein production was induced by adding IPTG to a final concentration of 1mM, and the culture was incubated overnight at 28°C. Subsequently, crude samples of induced and un-induced cells were analyzed on 12.5% SDS-PAGE (Figure 10). The SDS-PAGE results indicate that there is no overexpression of the c1 dioxygenase. On the coomassie stained gel there is a protein zone which is present at the induced samples, but not in the un-induced and which could be the dioxygenase. However, this protein zone has a molecular weight Figure 10. Comparison of protein expression at different transformed C43 bacterial clones after IPTG induction. Protein production was induced with 1mM IPTG. Cultures were incubated at 28°C overnight.
- 14. 14 of ~40kDa, which does not match the dioxygenase’s molecular weight of 29.9kDa. Thus, the following step was to pick up new colonies from the transformation plate. Five new clones were selected and inoculated in 5ml of LB medium with kanamycin (25μg/ml). The cultures were incubated at 37°C overnight, and the next day, flasks with 10ml of LB with 25μg/ml kanamycin were inoculated with 1/20 volume of the bacterial pre-culture and incubated at 37°C until the OD600 was 0.4 to 0.6. The protein production was induced by adding 1mM IPTG and the culture was incubated overnight at 28°C. A purified protein of known molecular weight, which was provided by lab members used as control. Crude samples of induced and un-induced cells were analyzed on 12.5% SDS-PAGE (Figure 11). The SDS-PAGE results, again, did not indicate overexpression of the c1 dioxygenase. On the coomassie stained gel there was no difference in protein zones between the un-induced and the induced samples. The cells maybe express the dioxygenase, but in very low concentration. Afterwards, the following experiment was to perform a new analysis with SDS-PAGE, in order to see comparatively whether BL21(DE3) or C43 cells express the dioxygenase. The analysis was performed with crude samples of IPTG induced C43 and BL21 (DE3) cells, which were electrophorated in a 12.5% SDS-PAGE, as described previously (Figure 12). Figure 11. Comparison of protein expression at different transformed C43 bacterial clones after IPTG induction. Protein production was induced with 1mM IPTG. Cultures were incubated at 28°C overnight.
- 15. 15 Results from the SDS-PAGE analysis did not show any dioxygenase overexpression neither from BL21(DE3) cells, nor from C43 cells. Conclusion Results from our analysis indicate that the clones of BL21 (DE3) and C43 cells that we tested did not overexpress the c1 dioxygenase. Within the framework of this 2 months rotation, we couldn’t continue the experiments and test more clones. In order to find a positive clone which would overexpress the dioxygenase, it is suggested as a future experiment to perform a colony PCR with specific primers for the amplification of the insert gene. In that way, it is possible to test many different clones and increase the possibilities to find a positive clone which will have the plasmid. Furthermore, when a positive clone will be found, it is recommended to test different conditions of bacterial growth and IPTG induction. For example, the temperature of bacterial growth, as well as, the final concentration of IPTG should be tested, in order to find the optimal conditions for dioxygenase overexpression. Figure 12. Detection of c1 dioxygenase in crude samples of induced C43 and BL21 (DE3) cells. Protein detection was achieved with Anti-His antibody. For positive control, it was used a purified protein of known molecular weight.
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