A hyperthermophilic bacteria Thermotoga maritima was used as a source of Beta-glucuronidase gene. Escherichia coli DH5Î± and E.coli strain BL21 (DE3) were used as mesophilic hosts, for the first step and second cloning step respectively and for expression of the cloned Beta-glucuronidase gene.
Cloning vector pTZ57R/T (Fermentas Inc., Hanover, Germany) was selected due to its fast cloning rate, high efficiency to yield about 90% recombinants,one step procedure, no additional modifications of PCR products required, ready-to-use vector (linearized and 3'-ddT tailed), MCS designed for easy manipulation with a cloned insert and blue/white screening. Plasmid pET-21a (Novogen Inc., Madison, WI, USA) was used as expression vector for getting high yield of recombinant gene.
Culturing was done for Escherichia coli DH5Î± and BL21 (DE3) in Luria Bertani (LB) medium
Yeast extract 0.5%
NaCl 1% in ddH2O in 250 ml Erlenmeyer flask
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For LB Agar medium,
Agar 1.5 % in above composition
autoclaved at 121Â°C and 15 lb/in2 pressure for 20 minutes.
Thermocycler (Biometra), gel casting unit, gel sealing tape, comb, gloves, microwave oven (LG), power supply devices, SYNGENE gel documentation system, Shaking Incubator at 37oC/22 oC (InnovaR 43 Incubator shaker series), Incubator at 37oC (ECOCELL), eppendorfs, centrifuge tubes, PCR tubes, Electrophoresis apparatus (WEALTES) ELITE 300 PLUS, water bath (VSB-30, wisebath DAIGGER incubator and companies inc. 620 lake view parkway, Vemons Hills. 1L 60061 USA), centrifuge machine (SIGMA laboratory centrifuges 3K30) at 40C, microcentrifuge machine (SANYO (MSE) Micro Centaur, 13000 rpm), Sonication Heat System (UP400S, ultrasonic Processor, utraschall Prozessor (Hielsher ultrasound, Technology), electric weight balance (Types AUW 2200 No D 450013067, SHIMODU Corporation Japan), vortex machine (My Lab TM SLV-6), spectrophotometer (CECIL CE 7200, 7000 Series), freezer -20oC (PEL Crystal Classic),Laminar flow cabinet (Technico Scientific Supply), Concentrator (RVC 2-25/Christ CT 02-05), Lyophilizer (Freeze dryer) and SDS-PAGE apparatus.
ISOLATION OF GENOMIC DNA
Genomic DNA was isolated according to Sambrook et al., 1989. 10 ml of Thermotoga maritimaa culture was used for the isolation of genomic DNA. The cells were then pelleted at 6500 rpm for 10 minutes at 4oC. The cell pellet was then gently suspended in 100 Âµl of TEN buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 10mM NaCl) and centrifuged at 6500 rpm for 5 minutes at 4oC. Supernatant was discarded and pellet was dried. Then cell pellet was dissolved in 100 Âµl SET buffer (50mM Tris-HCl pH 8.0, 50 mM EDTA, 25 % Sucrose) and 50 Âµl of freshly prepared lysozyme (10 mg/ml), followed by gentle mixing and incubation for 30 minutes at 37Â°C. After incubation, 100 Âµl 25 % sodium dodecyle sulphate (SDS) was added and tube inverted until lysis occurred. Then 500 Âµl of TEN buffer and 100 Âµl of 5 M NaCl was added. Equal volume of freshly prepared phenol-chloroform (1:1) was added and the suspension was centrifuged at 6500 rpm for 10 minutes at 4oC. The upper viscous phase was then drawn out with a micropipette, mixed with 1 volume of chloroform and subjected to centrifugation for at least 5 minutes at 6500 rpm at 4oC, after which upper phase was collected in a fresh tube and treated with chilled absolute ethanol to precipitate DNA, in a volume twice as present in the tube. The tube was placed overnight at -20 Â°C. DNA was centrifuged at 6500 rpm for 10 minutes in order to get a DNA pellet. The DNA was allowed to be air dried and finally dissolved in 50 Âµl TE buffer. 2 Âµl of RNase was added in this and incubated for 3 hours at 37oC, then stored at -20oC.
Agarose Gel Electrophoresis
1 % agarose gel solution was prepared by taking 0.5 g agarose, 1 ml of 50X TAE buffer (121 g Tris base, 28.6 ml glacial acetic acid, 50 ml 0.5M EDTA, pH 8.0 making volume up to 500 ml by distilled water) in 50 ml distilled water. The solution was heated to make it clear and transparent. 5 Âµl ethidium bromide (0.5 mg/ml) was added and the solution was poured in the gel caster and gel comb was fixed to make the wells. When the solution was solidified, gel comb was removed. The genomic DNA (calculated amount of the DNA in 5 ml of 10 X bromophenol blue) along with the l DNA HindIII digest marker was loaded in the wells of the gel. The gel was run at 80 volts, in 1X TAE buffer for 1 hour. After electrophoresis DNA bands were observed under UV light.
Quantification of DNA
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DNA quantification was done by visualizing on 1 % agarose gel as well as by spectrophotometeric calculations. DNA was loaded on 1 % agarose gel along with known amount of standard DNA marker and quantified under UV-light by comparing with the marker bands. DNA was also quantified spectrophotometrically by taking the absorbance at 260 nm using water as blank in BioSpec-1601 Shimadzu spectrophotometer. The quantity of DNA was calculated by applying the formula; Amount of DNA = 50 x OD at 260 nm x dilution factor where 1 OD at 260 nm = 50 Âµg of DNA. Purity of the DNA was checked by comparing the absorbance at 260/280 nm.
PCR Amplification of Thermotoga maritime beta-glucuronidase Gene
A pair of primer was designed based on the coding sequence of BglA from Thermotoga petrophila RKU-1 obtained from the NCBI server under the accession number CP000702 to carry the NdeI recognition sites upstream (underlined), sequence available in the NCBI database. These oligomers and Thermotoga petrophila RKU-1 chromosomal DNA was used to amplify the 1.341 kb fragment by employing the conditions of PCR cycling.
Gluco F 5'-CATATGAACGTGAAAAAGTTCCCTGAAGG-3'
Gluco R 5'-TCAATCTTCCAGACTGTTGCTTTTGACC-3'
PCR Amplification of Î²-glucosidase gene (BglA)
PCR amplification of Î²-glucosidase gene was done in a 20 Âµl reaction mixture with the components as:
Genomic DNA (40 ng/Âµl) 0.5 Âµl, Forward Primer (100 picomoles) 0.6 Âµl, Reverse Primer (100 picomoles) 0.6 Âµl, dNTPs (2.5 mM), 10X Taq buffer 02 Âµl, MgCl2 (25 mM) 02 Âµl, Taq Polymerase 0.5 Âµl, PCR water (dd H2O) 5.8 Âµl.
PCR Cycling Parameters
Fig. : PCR Amplification Cycle.
Thermocycler was set at following temperatures.
Denaturation 94Â°C --- 5 min.
*Denaturation 94Â°C --- 1 min.
*Annealing 52Â°C --- 1 min. (*35 cycles)
*Elongation 72Â°C --- 1 min.
Final Extension 72Â°C --- 20 min.
The final extension step was used to produce 3'-dA overhangs in PCR product to increase the efficiency of cloning and ligation.
Analysis of PCR product:
The PCR product was then analyzed on 1 % agarose gel after mixing with gel loading dye and loaded on the gel. The gel was run at 80 V for 90 minutes. The bands after visualizing in UV light were cut with the help of sterilized blade for purification.
Purification of PCR Amplified DNA Fragment from Agarose Gel
PCR product was gel purified by using QIA quick PCR purification kit (Qiagen, Hilden, Germany). Excised PCR amplified DNA fragment from agarose gel was transferred to a pre-weighed empty eppendorf tube. The eppendorf tube containing the gel slices was weighed again. Weight of the gel slices was calculated by subtracting the weight of empty eppendorf tube. To 100 mg of gel 300 Âµl of QG (gel melt solution) was added and incubated at 50Â°C for 10 minutes with vortexing of the tube every 2-3 minutes during incubation. A QIA quick spin column was placed in a 2 ml collection tube and dissolved gel solution was added and centrifuged at 12000 rpm for 30 seconds. After discarding the flow through 650 Âµl of PE buffer (wash buffer) was then added and centrifuged at high speed for 30 seconds. The column was placed into a clean 1.5ml microcentrifuge tube. To elute DNA, 50 Î¼l of pre-warmed (50Â°C) EB buffer (10 mM Tris-Cl, pH 8.5) was added and incubated at 50Â°C for 3 minutes followed by centrifugation at 12000 rpm for 1 minute. Then the second elution was performed, cleaned PCR amplified DNA fragment was stored at -20Â°C.
CLONING OF BETA-GLUCOSIDASE GENE (BglA) IN pTZ57R/T
pTZ57R/T Cloning Vector
TA cloning system used pTZ57R/T as cloning vector for cloning of PCR products with 3'-dA overhangs. Taq DNA polymerase produced these overhangs (Fig.). The vector has been pre-cleaved with Eco32I (an isoschizomer of EcoRV) and treated with terminal deoxynucleotidyl transferase to create 3'-ddT overhangs at both ends. When a PCR fragment with 3'-dA overhangs is ligated into the vector, a circular molecule with two nicks (Fig.) was produced.
Fig: Restriction map of vector pTZ57R/T (Clark, J. M., 1988).
Ligation of purified Beta-glucosidase gene (BglA) in pTZ57R/T
The purified PCR amplified Î²-glucosidase (BglA) was quantified and ligated into pTZ57R/T using T4 DNA ligase. The 30 Âµl ligation reaction mixture contained: 1X ligation buffer (40 mM Tris-HCl pH 7.8 at 25 Â°C, 10 mM MgCl2, 10 mM DDT and 5 mM ATP), 1X polyethylene glycol (PEG 4000) solution (5 % w/v), 20 Âµg BSA, 155 ng cut pTZ57R/T vector, ~350 ng Î²-glucosidase DNA and 5 units of T4 DNA ligase. The ligation mixture was incubated overnight at 22Â°C. Control, containing the circular plasmid without insert was also incubated parallel with the experimental ligation mixture. The circular product was used directly to transform E.coli DH5Î± cells with high efficiency. The 3'-ddT overhangs at both ends of the cloning site which add extra adenines to the ends of PCR products prevent re-circularization of the vector during ligation, therefore resulting in high cloning yields.
Fig.2. Ligation of PCR fragment in the pTZ57R/T vector.
Preparation of Competent Escherichia coli Cells
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Competent cells were prepared as described by Maniatis et al., (1989). A single colony of cells was inoculated into 5 ml of LB medium (1 % tryptone, 1 % NaCl and 0.5 % yeast extract) and grown overnight at 37oC and 200 rmp. About 1 ml of overnight grown culture of Escherichia coli DH5Î± was inoculated into 100 ml of LB medium and incubated in a shaking incubator at 37Â°C for 3 hours. Then the cells were collected in 50 ml falcon tube, cooled for 15 minutes and centrifuged in a pre-cooled centrifuge at 6000 rpm for 10 minutes at 4Â°C. The supernatant was decanted off and the cell pellet was suspended in 20 ml ice-cold 50 mM CaCl2 and incubated on ice for 40 minutes. The cells were centrifuged at 6000 rpm for 10 minutes at 4Â°C. Again the supernatant was decanted off and the cell pellet was resuspended in 2 ml ice-cold 50mM CaCl2 and kept on ice for 30 minutes. 200 Âµl of these cells were streaked on LB-Ampicillin plate to check the contamination of the cells. Competent cells were stored at 4oC and used with in two or three days for transformation.
Transformation of E.coli (DH5Î±) Cells With Î²-glucosidase/pTZ57R/T
E.coli DH5Î± cells were transformed according to standard and recommended procedures (Sambrook and Russell, 2001). A 200 Âµl of competent cells of E.coli DH5Î± were briefly thawed, mixed rapidly with 15 Âµl of the ligation reaction mixture along with a control containing circular plasmid without insert and incubated on ice for 40 minutes. The cell/plasmid mixtures were heat shocked at 42Â°C for 90 seconds and quickly placed on ice for further 5 minutes. LB medium (800 Âµl) was added to each mixture, mixed and incubated for 1 hour at 37Â°C in an incubator. Bacterial cells were then spread on LB plates containing the required antibiotics for the selection of recombinants.
Blue-White Recombinants Colonies Selection
Selective LB Petri-plates for blue white screening of recombinant, were prepared by using 100 ml LB agar medium containing 135 Âµl of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal 40 mg/ml), 200 Âµl of ampicillin (25 mg/ml) and 130 Âµl of 0.1 mM isopropyl-Î²-D-thiogalactopyranoside (IPTG). The transformed cells were spread onto the LB agar plates which were incubated overnight at 37Â°C. After the appearance of blue and white colonies on these plates the transformed white colonies were selected.
Direct analysis of positive transformation was carried out by colony PCR. White colonies were selected from plate for colony PCR. The Î²-glucosidase gene (BglA) cloned in pTZ57R/T was amplified by polymerase chain reaction (PCR) using forward and reverse primers. Amplification reaction mixture prepared, single colony was picked from plate and transferred to the PCR reaction mixture. The reaction mixture was incubated in a thermocycler, the colony PCR amplification was then examined on 1 % agarose gel. For colony PCR following reaction components are required.
The 25 Âµl amplification reaction mixture contained
Forward Primer (100 picomoles) 0.6 Âµl, Reverse Primer (100 picomoles) 0.6 Âµl, dNTPs (2.5 mM) 08 Âµl, 10X Taq buffer 02 Âµl, MgCl2 (2.5 mM) 02 Âµl, Taq Polymerase 0.5 Âµl, PCR water (dd H2O) 5.8 Âµl.
PCR Cycling Parameter
Thermocycler was set at following temperatures.
Denaturation 94Â°C --- 5 min
*Denaturation 94Â°C --- 1 min
*Annealing 52Â°C --- 1 min (*35 cycles)
*Elongation 72Â°C --- 1 min
Final Extension 72Â°C --- 20 min
Plasmid DNA Isolation
Plasmid isolation was carried out by alkaline lysis method, with a modification of the methods of Birnboim and Doly (1979). A single bacterial colony containing the plasmid of interest (detected by colony PCR) was inoculated into 5 ml of LB medium containing 10 Âµl ampicillin (25 mg/ml). The culture was incubated overnight at 37oC at 100 rmp. About 3 ml of the overnight grown culture was centrifuged at 6000 rmp (6,700 Ã- g) for 5minutes at 4oC. The cell pellet was resuspended in 100 Âµl of ice-cold Solution I (prepared by dissolving 50 mM glucose, 25 mM Tris-HCl and 10 mM EDTA, pH 8.0) and placed on ice for 10 minutes. Then 200 Âµl of freshly prepared Solution II (0.2 N NaOH and 1% SDS, pH 12.0) was added. The tube was closed tightly and the contents were mixed by inverting the tube rapidly five times so that the entire surface of the tube came in contact with Solution II. The tube was incubated in ice for 5 minutes and 150 Âµl of ice-cold Solution III (prepared by mixing 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid and 28.5 ml of H2O) was added. The mixture was incubated in ice for 10-15 min and then centrifuged at 12000 rmp (13,400 Ã- g) for 5 minutes at 4oC. The supernatant was transferred to a fresh tube. Phenol/chloroform extraction was performed by adding equal volume of phenol:chloroform (1:1) in the above material and mixed followed by centrifugation at 12000 rmp for 2 minutes at 4oC. The supernatant was transferred to a fresh tube. The plasmid DNA was precipitated with 2 volumes of ice cold absolute ethanol. The mixture was allowed to leave on ice for 10-15 minutes and then centrifuged at 12000 rmp (13,400 Ã- g) for 15 minutes at 4oC. The supernatant was removed and the pellet of plasmid DNA was rinsed with 100 Âµl of 70 % ethanol. The supernatant was removed after centrifugation at 12000 rpm for 5 min and the pellet was allowed to dry in the air until transparent. The DNA was dissolved in 50 Âµl of TE (pH 8.0) buffer, the DNA was incubated for 2-3 hours at 37oC after adding 1 Âµl of RNase. The sample was electrophoresed on 1% agarose gel and analyzed under UV-light. The plasmid DNA was stored at -20oC.
Restriction analysis of Î²-glucosidase (BglA) in pTZ57R/T
Cloning of BglA in pTZ57R/T was confirmed by double restriction analysis of the recombinant plasmid with specific restriction enzymes. Double restriction digestion of the recombinant plasmid was carried out with NdeI and HindIII in 2X tango buffer (Fermentas) in which both the enzymes had optimal activity. For double restriction the 20 Âµl reaction mixture containing ~ 1 Âµg of the recombinant plasmid, 10 units of NdeI in 2X yellow tango buffer (66 mM Tris acetate pH 7.9 at 37 Â°C, 20 mM Magnesium-acetate, 132 mM Potassium-acetate and 0.2 mg/ml BSA), was incubated overnight at 37oC. After the overnight incubation, HindIII (10 units) was added into it and incubated at 37Â°C for another 3 hours. The samples were electrophoresed on 1% agarose gel and analyzed under UV-light.
Sequencing of Î²-glucosidase gene (BglA)
After confirmation of the cloning in pTZ57R/T by double restriction, the sequencing of the insert was performed and the sequences were determined in both directions by vector specific T3 and T4 primers.
DNA sequencing (Purification of the recombinant plasmid)
The recombinant plasmid containing the gene of interest was isolated from 5 ml culture using QIA prep spin miniprep kit (QIAGEN, Hilden, Germany). A single colony was inoculated in 5 ml of LB medium and grown overnight at 37oC and 100 rpm. The overnight growth culture was centrifuged at 12000 rpm for 2 minutes at room temperature and the resulting pellet of the bacterial culture was resuspended in 250 Âµl of buffer PI (50 mM Tris HC1 pH 8.0, l0 mM EDTA, 10 Âµg/ml RNase A) followed by the addition of 250 Âµl of Buffer P2 (200 mM NaOH, 1% SDS, 1M NaOH (200 ml), 10% SDS (100 ml), H2O (700 ml). The contents of the tube were mixed by inverting the tube gently until the solution become viscous and slightly clear. Then 350 Âµl of Buffer N3 (100 mM Tris-HC1 pH 6.3, 15 % ethanol, 0.15 % triton, 900 mM KC1) was added and mixed gently by inverting the tube 4-6 times. The sample was centrifuged at 12000 rpm for 10 minutes at room temperature. The pellet was discarded and the supernatant was loaded to the QIA prep spin column. The sample was centrifuged for 1 min and the flow through was discarded. The QIA prep spin column was washed by adding 0.75 ml of Buffer PE (10 mM Tris-HC1 pH 8.3, 50 mM KC1, 1.5 mM MgCl2) and centrifuged for 1 minute. The flow through was discarded and the column was centrifuged for an additional 1 minute to remove residual wash buffer. To elute DNA, QIA prep spin miniprep column was placed in a clean 1.5 ml microcentrifuge tube and 50 Âµl of TE buffer was added to the column. The column was allowed to stand for 1 minute and centrifuged for 1 minute at 12000 rpm. The quality and quantity of the purified DNA was determined by 1% agarose gel electrophoresis.
Sequence data were analysed by examining nucleotide and aminoacid sequence analysis and theoretical molecular weight determination by using program of Expasy ProtParam (Gasteiger et al., 2005). Database homology searches were performed by using Basic Local Alignment Search Tool program (Altschul et al., 1990). The amino acid sequence of Î²-glucosidase was compared with its homologues from bacteria by using programe of CLUSTAL W/ DDBJ (Thompson et al., 1994).
CLONING OF Î²-GLUCOSIDASE (BglA) IN pET-21a
Ligation of BglA gene in pET-21a
After confirming the sequence, the recombinant pTZ57R/T containing Î²-glucosidase gene (BglA) was restricted with NdeI and HindIII. Expression vector pET-21a (+) was also cut with the same two enzymes. The plasmids were first restricted with NdeI and then HindIII was added to the same restriction mixture. In each case the 30 Âµl restriction mixture contained 10 Âµlof ~2Âµg plasmid pET-21a (+) and 1Âµlof 10 units NdeI in 3 Âµl of 2X tango buffer. The reaction mixture was incubated at 37Â°C for overnight. After overnight incubation l Âµl of 10 units HindIII were added to the same reaction mixture and incubated at 37Â°C for another 3 hours. The restricted fragment as well as the double digested pET-21a (+) vector were electrophoresed on 1% agarose gel, purified from the gel and ligated together. The 30 Âµl ligation reaction mixture contained: 1X ligation buffer (40 mM Tris-HCl pH 7.8 at 25Â°C, 10 mM MgCl2, 10 mM DDT and 5 mM ATP), 1X polyethylene glycol (PEG 4000) solution (5% w/v), 20 Âµg BSA, ~100Âµg double digested pET-21a, ~350 ng Î²-glucosidase gene and 5 units of T4 DNA ligase. The ligation mixture was incubated overnight at 22Â°C.
Transformation of E.coli (DH5Î±) Cells With Î²-glucosidase/ pET-21a
A 200 Âµl of competent cells were added to 30Âµl of the ligation reaction mixture and one control containing circular plasmid. Mixed gently, and incubated on ice for 40 minutes. The cell/plasmid mixture was heat shocked at 42oC for 90 seconds and quickly placed on ice for further 5 minutes. Liquid LB medium (800 Âµl) was added to each mixture, mixed and incubated for 1 hour at 37oC incubator. The medium was centrifuged and the cells (200 Âµl) were spread on LB agar supplemented with ampicillin (25 mg/ml ampicillin). Plates were incubated at 37oC overnight. The positive clones were inoculated in to 5 ml of LB medium and their plasmid isolation was done on small scale as stated elsewhere.
Direct analysis of positive transformation was carried out by colony PCR. Colonies were selected from plate for colony PCR. The Î²-glucosidase gene (Bgl) cloned in pET-21a was amplified by polymerase chain reaction (PCR) using forward and reverse primers. The 20 Âµl amplification mixture reaction contained: 1X PCR buffer (75 mM Tris-HCl pH 8.8 at 25Â°C, 20 mM (NH4)2SO4 and 0.01 % Tween 20), 0.2 mM dNTP mixture, 2.5 mM MgSO4, 100 pmoles of each primer and 2.5 units of Taq DNA polymerase. Single colony was picked from plate and transferred to the reaction mixture. The reaction mixture was incubated in a thermal cycler at 94Â°C for 5 minutes and then underwent 35 amplification cycles of 1 minute at 94Â°C, 1 minute at 52Â°C and 1 minute at 72Â°C, followed by a final incubation of 20 minutes at 72Â°C. The colony PCR amplification was then examined on 1% agarose gel. For colony PCR following reaction components are required.
Forward Primer (100 picomoles) 0.6 Âµl, Reverse Primer (100 picomoles) 0.6 Âµl, dNTPs (2.5 mM) 08 Âµl, 10 X Taq buffer 02 Âµl, MgCl2 (2.5 mM) 02 Âµl, Taq Polymerase 0.5 Âµl, PCR water (dd H2O) 5.8 Âµl.
Restriction Analysis of Î²-glucosidase (BglA) in pET-21a
Cloning of Î²-glucosidase gene (BglA) in pET-21a (+) was checked by double restriction of the recombinant plasmid with specific restriction enzymes. The double restriction was carried out with HindIII and NdeI in 1X tango buffer. For double restriction, the 30Âµlreaction mixture contained ~1 Âµg of the recombinant plasmid, l Âµl of 10 units of HindIII and 10 units of NdeI in 3 Âµl of 2 X tango buffer. The reaction mixture was incubated at 37 Â°C for 3 hours. After the required incubation period, the samples were loaded on 1 % agarose gel and the restriction was analyzed under UV-light.
Restriction map of pET-21a
Fig: pET-21a expression vector (restriction map).
Plasmid DNA Isolation
Plasmid isolation was carried out by alkaline lysis method. A single bacterial colony containing the plasmid of interest (detected by colony PCR) was inoculated in to 5 ml of LB medium containing 10 Âµl ampicillin (25 mg/ml). The culture was incubated overnight at 37oC at 100 rmp. From 3 ml overnight grown culture plasmid was isolated as stated elsewhere. The DNA was dissolved in 50 Âµl of TE (pH 8.0) and 1 Âµl of RNase was added, mixture was incubated for 2-3 hours at 37oC. After the incubation period, the sample were electrophoresed on 1 % agarose gel and analyzed under UV-light. The plasmid DNA was stored at -20oC.
Expression and Isolation of Î²-glucosidase (BglA) in E.Coli BL21
E. coli BL21 (DE3) competent cells were prepared and transformed with purified recombinant pET 21a harbouring Î²-glucosidase gene. Single colony from LB agar ampicillin (25 mg/ml) plate was used to inoculate 10 ml of LB medium containing ampicillin at a final concentration of 25 mg/ml in 100 ml flask and allowed to grow overnight at 37Â°C with vigorous shaking. The overnight grown culture was then added to two flasks of 100 ml LB media containing 200 Âµl ampicillin (25 mg/ml) and grown at 37Â°C until its OD was reached 0.6 and 0.8 respectively at 600nm. The cultures were induced with 0.5 mM isopropyl D-thiogalactopyranoside (IPTG) and incubated for another 24 hours at 22Â°C. Cells were harvested by centrifugation at 6000 rpm for 10 min. The resulting supernatant was used as the source of extra cellular enzyme. The cell pellet was re-suspended in 500 Âµl of 50 mM Tris-HCl (pH 8.0) and cells were disrupted by sonication on ice until cells were broken, using sonicator UP 400S at 60amplitude for 20 cycles. The mixture was centrifuged at 12000 rpm for 10 minutes, the supernatant was used as cell extract. The cultures were allowed to continue for 48 and 72 hours incubation. The same procedure was repeated for the extraction of crude enzyme. Crude supernatant and cell lysate samples were run on 12 % SDS gel. Give heat treatment to the crude supernatant and cell lysate at 80oC for 45 min; run these samples on 12% SDS gel. BL21 (DE3) containing pET-21a without insert was used as a control for comparison of expression on SDS-PAGE.
Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to check the expression of protein and for the determination of molecular weight. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) 12% slab gels were prepared using a modification of the procedure described by Laemmli, (1970). A protein gel casting system was employed in the preparation of gels using 12 cm x l cm glass plates. The gel assembly was prepared by placing spacers in-between the sides of the two glass plates and holding them with the help of clamps. Once the gel casting system was set up, the first five components of the 12 % resolving gel mixture were mixed. TEMED was added last to initiate the gel polymerization and the solution rapidly poured between the glass plates leaving space of about 1.5 cm for stacking gel. The 12 % resolving gel was left undisturbed to allow the gel to polymerize. Once the gel had set, 5 % stacking gel, prepared as for the resolving gel, was poured on the top of the resolving gel and a plastic comb inserted between the plates to form 30 Âµl wells for loading of analytical samples.
Solutions Used in Running of SDS-PAGE
The following reagents used for SDS-PAGE.
Running Buffer, 1X Tris-glycine buffer (pH 8.3) was prepared by dissolving 3.0 g of Trizma base, 18.7g of glycine and 1 g of SDS in distilled water; adjusting the pH to 8.3 with NaOH/HCl before making the volume up to 1 L. Solution was stored at room temperature.
30 % acrylamide/bisacrylamide solution was prepared by dissolving 29 g of acrylamide and 1 g bisacrylamide in 100 ml of distilled water. The solution was filtered and stored at 4oC.
Staining solution was prepared by dissolving 0.1% of Coomassie brilliant blue R-250 in acetic acid: methanol: distilled water (10:40:50). 0.1% of Coomassie brilliant blue R-250 dissolved in 220 ml of methanol and 50 ml of glacial acetic acid, volume was made up to 500 ml with distilled water.
Distaining solution was prepared by mixing 150 ml methanol, 50 ml acetic acid and volume was made up to 500 ml with distilled water. Gel stored solution was prepared by mixing 50 ml of the acetic acid to 450 ml of distilled water.
2 X gel loading buffer (pH 6.8^) or Loading dye was prepared by mixing 4 % SDS, 20 % glycerol, 0.2 % bromophenol blue dye, 200 mM Dithiothreitol (DTT) (Sigma Chemical Co. USA) in 1 ml of 100 mM Tris-HCl (pH 6.8) and 7 ml of distilled water in a glass vial and stored at 4oC. Before use 0.5 ml Î²-mercaptoethanol was added for samples preparation.
Resolving buffer (1.5 M Tris pH 8.8) was prepared by dissolving 36.3 g Tris in distilled water, pH was adjusted to 8.8 and volume was made up to 200 ml with distilled water.
Stacking buffer (0.5 M Tris pH 6.8) was prepared by dissolving 12.1 g Tris in distilled water, pH was adjusted to 6.8 and volume was raised to 200 ml distilled water.
12 % Resolving gel (10 ml) was prepared by mixing 3.3 ml of distilled water, 4.0 ml of 30 % acrylamide/bisacrylamide solution, 2.5 ml of 1.5 M Tris-HCl (pH 8.8), 0.1 ml of 10 % SDS, 0.1 ml of 10 % ammonium persulphate and 0.004 ml of N,N,N/,N/-Tetramethylethylenediamine (TEMED). Freshly prepared resolving gel was poured inside the gel assembly leaving 0.5 inches vacant space at the top. Almost 100Âµl of distilled water was layered at the top of the gel to give a flat surface and to remove oxygen, which may inhibit polymerization. The gel was allowed to polymerize for 30 minutes.
5% Stacking gel (5 ml) was prepared by dissolving 3.075 ml of distilled water, 0.670 ml of 30 % acrylamide/bisacrylamide solution, 1.25 ml of 0.5 M Tris-HCl (pH 6.8), 0.05 ml of 10 % SDS, 0.025 ml of 10 % ammonium persulphate and 0.005 ml of TEMED. Water was removed and 5 % stacking gel was poured in the gel assembly followed by insertion of comb. After polymerization gel comb was taken out and wells were washed with Tris-glycine buffer by means of a syringe. Gel assembly was settled on the gel chamber after removing the bottom spacer.
Protein samples (5-10 Âµl) containing loading dye (5-10 Âµl) was prepared by heating in boiling water for 3 minutes. Protein markers, protein extract from crude broth, sonicated samples and control were loading on the gel, electrophoresed at a constant voltage of 150 V.
The cassette containing the set gel with the well forming comb removed was clamped into the electrophoresis apparatus containing 1X Tris-glycine buffer in its bottom reservoir. The upper reservoir was also filled with the same buffer. 10 - 20 Âµl of the protein samples prepared above were loaded into the wells formed in the stacking gel with a fine tip. Samples were electrophoresed at a constant voltage and electrophoresis performed for about 2- 3 hours until the bromophenol blue dye reached the bottom of the resolving gel. The current was then turned off and the gel was removed from the glass plates of the cassette.
Staining of SDS Gel
When gel was run completely, it was placed in an enamel tray containing Coomassie brilliant blue staining solution for 30 minutes with constant agitation. After staining, gel was placed in the destaining solution again with constant agitation until the background of the gel became transparent and protein bands were visible. Gel was stored in 10 % acetic acid.
RECOMBINANT BETA-GLUCOSIDASE ACTIVITY ASSAY
Beta-glucosidase activity was assayed at 90Â°C using 4 mg/ml p-nitrophenyl-Î²-D-glucopyranoside (pNPG) as substrate in 50 mM sodium citrate buffer (Mcllvaine Buffer) of pH 7.0. The reducing sugars released were quantified by the p-nitrophenol method according to Miller, (1959).
Reagents Preparation for Enzyme Assay
The different reagents used in the enzyme assay procedure which was as follow
Substrate: 4 mg p-nitrophenyl-Î²-D-glucopyranoside (pNPG) used as substrate in 1ml of sodium citrate buffer (Mcllvaine Buffer) of pH 7.0.
Mcllvaine Buffer/Sodium citrate buffer (pH 7.0): For the Mcllvaine Buffer, stock solutions of 0.1 M citric acid (21.01 g/l) and 0.2 M Na2HPO4 (35.61 g/l) were prepared. Then 18 ml of 0.1 M Citric acid was mixed with 82 ml of 0.2 M Na2HPO4 and the pH was adjusted to 7.0 with 0.1 M citric acid if found necessary.
1 M Sodium Carbonate Solution: For the preparation of 1 M Na2CO3 solution 105.99g of sodium carbonate was dissolved in 1 L distilled water.
Activity Assay Method
200 Âµl of substrate, 390 Âµl of buffer along with 10 Âµl of crude enzyme (supernatant) were added in clean test tubes and incubated at 90oC for 10 minutes. After incubation, 3 ml of 1 M Na2CO3 was added in each test tube to stop the reaction. Then the absorbance was measured at 405 nm. To estimate the extent of reducing sugar in the broth and to eliminate its absorbance value in the test, blank was made, without incubation for 10 minutes at 90oC, by using 200 Âµl of substrate, 390 Âµl of buffer of pH 7.0, 3 ml of 1 M Na2CO3 and 10 Âµl of enzyme, take its absorbance at 405 nm was set to auto zero. The reducing sugars released were measured by using p-nitrophenol (pNP) as standard.
Standard Curve of Para-Nitrophenol (pNP)
Para-nitrophenol (pNP) was used as a standard for enzyme activity. A calibration curve was established with p-nitrophenol (pNP) to convert spectrophotometer reading into ÂµM of p-nitrophenol (pNP). The stock solution contained 1 mM p-nitrophenol (0.0139 g/100 ml) was serially diluted ranging from 20 ÂµM to 500 ÂµM. Then 3 ml 1 M Na2CO3 was added in each dilution. Blank was made with only distilled water. The absorbance was measured at 405 nm on spectrophotometer. The following table shows all the data and absorbance.
Distilled Water (Âµl)
Table: Absorbance values of different concentration of Para-nitrophenol (ÂµM).
Figure: Standard Curve of Para-nitrophenol.
DETERMINATION OF SPECIFIC ACTIVITY
Specific activity of enzyme is defined as the units of enzyme/mg of protein.
Estimation of Protein
Soluble proteins in the culture supernatant and in the purification operation were estimated by Bradford dye-binding method (1976). Protein concentration was determined by Lowry et al., (1951) with Bovine serum albumin (BSA) (Sigma Chemical Co. USA) as a standard.
Bradford's Reagent: For dye binding reagent preparation, 100 mg of Coomassie brilliant blue G-250 was dissolved in 50 ml of 95 % ethanol and then 100 ml of 85 % phosphoric acid was added to it. The solution was mixed and diluted to1 liter with distilled water and filtered twice. Final concentration in the reagent was 0.01 % w/v Coomassie Brilliant Blue G-250, 4.7 % ethanol and 8.5 % w/v phosphoric acid.
Method for protein estimation: 5 ml of dye binding reagent was added to 10 Âµl of cell lysate along with 990 Âµl of distilled water. In the case of supernatant 100 Âµl of crude enzyme and 900 Âµl of distilled water was used. The reagent blank was prepared by taking 1 ml of water and 5 ml of reagent. Absorbance was measured at 595 nm after two minutes against the reagent blank and concentration was estimated from the standard curve.
Standard Curve for Protein Estimation
A standard curve was prepared by using the stock solution of BSA which was prepared by dissolving 0.1 g of the albumin in 100 ml of distilled water. Appropriate dilutions containing 10-200 Âµg/ml of BSA were made. Then 5 ml of Bradford reagent was added to 1ml of the standard albumin solution and absorbance was measured at 595 nm after two minutes against the blank reagent. The blank reagent was prepared by mixing 1ml of distilled water and 5 ml of dye binding reagent. The standard curve was plotted between absorbance and concentration of the BSA.
Concentration of BSA Stock (Âµg/ml)
Table: Absorbance at different concentration of BSA.
Figure: Standard curve for protein estimation by Bradford method.
"One unit of enzyme activity is defined as "The amount of enzyme which liberates 1 Âµmol of reducing sugar per minute. Specific activity was expressed as unit per milligram of protein.
The enzyme activity was then converted in to U/ml by applying the following formula:-
Enzyme activity = Units/ml = Concentration from graph x dilution factor x 1000 Time of incubation x 400
Specific activity = Units/mg of protein = Units/ml of enzyme
mg of protein/ml enzyme
CHARACTERIZATION OF RECOMBINANT BETA-GLUCOSIDASE
The recombinant Î²-glucosidase was characterized for optimal pH, optimal temperature, thermostability, molecular mass and the effect of various metal ions on the Î²-glucosidase activity. The molecular mass of Î²-glucosidase was determined by SDS-PAGE.
Optimization of pH for Î²-glucosidase activity
The optimum pH of BglA activities were measured at pH ranging from 3.0 to 11.0, in increment of 1.0 pH unit, under the assay conditions at 80oC for 10 minutes. Using 50 mM sodium citrate buffer (Mcllvaine Buffer) in a range from pH 3.0 to 7.0, HEPES (pH 8.0) adjust pH with 1 M HCl and 3-[cyclohexylamino]-1-propanasulfonic acid (CAPS) buffer (pH 9.0-11.0) adjust pH with 1 M NaOH. For assay, 20 Âµl of enzyme, 200 Âµl of substrate along with 380 Âµl of buffer was incubated at 90oC for 10 minutes. After incubation 3 ml of 1 M Na2CO3 was added in each test and then absorbance was measured at 405 nm.
Optimization of temperature for Î²-glucosidase activity
The optimum temperature was determined by standard assay at temperature ranging from 30-110oC in 50 mM sodium citrate buffer (Mcllvaine Buffer) pH 7.0. For assay, 20 Âµl of enzyme, 200 Âµl of substrate along with 380 Âµl of buffer was incubated at various temperatures for 10 minutes. After incubation 3 ml of 1 M Na2CO3 was added in each test tube and the absorbance was measured at 405 nm.
The temperature stability was determined by incubating crude supernatant enzyme sample at 30-100oC. The incubated enzyme sample was withdrawn at different time interval, and then the residual enzyme activity was determined at 90oC in sodium citrate buffer of pH 7.0. For assay, 20 Âµl of enzyme, 200 Âµl of substrate along with 380 Âµl of buffer was incubated at 90oC for 10 minutes. After incubation 3 ml of 1 M Na2CO3 was added in each test and then absorbance was measured at 405 nm.
Effect of metal ions concentration
To check the effect of various Metal ions on Î²-glucosidase activity, the enzyme was incubated in the presence of 1 mM solution of MnCl2, CaCl2, CdCl2. H2O, HgCl2, MgSO4, Pb(NO3)2, ZnCl2, CoCl2, FeCl3, NiCl2, CuSO4 and metal chelator such as EDTA for 10 minutes at 90oC. For assay 360 Âµl of sodium citrate buffer of pH 7.0, 20 Âµl of metal solution, 200 Âµl of substrate with 20 Âµl of enzyme were incubated at 90oC for 10 minutes followed by the addition of 3ml of 1 M Na2CO3 to stop the reaction. The absorbance was measured at 405 nm to measure the residual activity.