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E. coli was grown in Luria-Bertani broth (LB) at 37°C. Ampicillin (100μg/ml) and kanamycin (100μg/ml) were added to broth or agar as needed. When necessary, IPTG (isopropyl-β-d-thiogalactopyranoside) (0.1 mM) and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (20μg/ml) were spread on agar plates 30 min prior to plating. The strains, plasmids, and primers used for this study are listed in Table 4.1. The work was done aseptically under the laminar air flow workstation.
Table4.1 Bacterial strains, plasmids, kits, restriction enzymes and primers used in this work
Strain, plasmid, or primer
Genotype, description, or sequence
Reference or source
F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)
F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ-
MinElute Gel Extraction (Cat. no. 28604)
N-terminal His•Tag/thrombin/T7•Tag, origin of replication: f1, pBR322
Total genomic DNA from E.coli was isolated using kit RKT 11 from Chromous Biotech. Took 1.5ml of overnight grown E.coli culture in a 2ml vial.Spin down the culture at 13000 rpm for 2 min.To the pellet added 750 µl of suspension buffer. Broke the pellet by vortexing and added 5 µl of RNase solution. Placed thevial at 650C for 15 mins.After incubation, added 1ml lysis buffer, vortexed for a minute and placed the vial at 650C for 15 mins. Spun the sample at 13000 rpm for 2 min at room temperature. Decanted the clear supernatant into two 2 ml fresh vials (900 µl each), added 900 µl of isopropanol to each of the above vials and mixed well.Spun at 13000 rpm for 15 mins at room temperature. Discarded the supernatant.To the pellet, added 1 ml of 70% ethanol. Spin at 13000 rpm for 15 min at room temperature. Discarded the supernatant.Repeated the last step.Dried the pellet at 370C.Added 50 µl of glass distilled water to each of the vials. Suspended the DNA by placing the vials at 650C for 15 min.Stored at -210C.
Determination of yield: -
1% Agarose gel electrophoresis was carried out.For a 1% agarose gel, weighed out 1g of agarose into a flask and added 100ml of 1 x TAE.Heated the solution on a hot plate until agarose is completely dissolved.Allowed it to cool in a water bath set at 50 - 55 °C for 10 min.Prepared gel-casting tray by sealing ends of gel chamber by using gel casting system. Placed a gel comb in the gel tray.Added 5 ul of ethidium bromide to cooled gel and poured into gel tray. Allowed it to cool for 15-30 min at room temperature.Removed comb, placed in electrophoresis chamber and covered with 1x TAE buffer.Added loading buffer to samples on a parafilm strip.Loaded DNA and standard 1kb DNA ladder onto gel.Electrophoresed at 100V for 1 h.Visualized DNA bands using gel-imaging system (fig 4.4).
4.3 PCR amplification of murA gene of E. coli.
Genomic DNA of E. coli was used for amplification of murA gene using specific modifiedprimers stated in the table 4.1. PCR mixture was prepared as given in table 4.2. PCR cycles setup is stated in table 4.3. PCR product was subjected to 1% gel electrophoresis with 1kb DNA ladder (Fig.4.1)
Table 4.2: PCR Mix
Table 4.3: PCR Cycle setup
4.4 Digestion and purification of plasmid vector
Plasmid vector, pET-28c, was digested with XhoI and NdeI and purified(Fig.: 4.3)
4.5 Digestion of murA
The murA digestion was carried out using the digestion mix composition stated in table 4.4. Composition was made accordingly and was left overnight at 370C for the proper digestion.
Table 4.4: murA Digestion mixture composition
4.6Purification of double digested murA
The double digested murA was purified using Qiagen purification kit. 45µl PCR product was added with 250 µl PBI and mixed.Loaded it on the column.Centrifuged at 8000 rpm for 1 min.Discarded the supernatant.To the column, added 500µl wash buffer (PE).Centrifuged at 10,000 rpm for 1 min.Placed column in a 1.5 ml microcentrifuge tube.Elution of DNA was done by adding water in the center of membrane and allowing it to stand for 1 min followed by centrifugation at 10,000 rpm for 1 min.Purified product was subjected to 1% agarose gel electrophoresis with non-purified PCR product.
4.6 Ligation of double digested murA and pET-28c
Ligation mixture was prepared according to the composition given in table 4.5. The mixture was subjected to overnight incubation at 160C for proper ligation.
Table 4.5: Ligation mixture composition
4.7 Preparation E.coli competentcells
Competent cells of E.coli were prepared using standard calcium chloride method. Inoculated a single colony of E.coli in 20ml of LB broth and icubated overnight at 370C. Inoculated 1% of this culture in 200ml LB broth. After 1 ½ hours of incubation, set the OD to 0.3 at 600nm. Culture, tips, MCTs, water, 0.1 M CaCl2, and 50% glycerol were kept on ice prior to proceeding further. Culture was drawn in a 50 ml centrifuge tube and centrifuged at 4,000 rpm for 10 mins. at 40C. The formed pellet was then resuspended in distilled water and centrifuged again at 4,000 rpm for 10 mins. at 40C. Resuspened the pellet in 0.1 M CaCl2 and centrifuged at 4,000 rpm for 10 mins. at 40C. Pellet was resuspended in 10ml of 0.1 M CaCl2and kept on ice for 1 ½ hours. Stored at -200C.
4.8 Transformation of competent E.coli cells
Competent E.coli cells were transformed with the plasmid vector carrying murA insert using heat shock method.Vial of competent cells from -200C were thawed on ice before proceeding further. Added 5 µlof ligation mixture into the cells. Kept it on ice for 10 mins. Gave the cells a 'Heat Shock' of 420C for exactly 90 secs. Added 1 ml of LB broth and incubated at 370C for 45 mins. Centrifuged at 6,000 rpm for 10 mins. at room temperature. Discarded the supernatant except around 150-200 µl.Resuspended the pellet in remaining supernatant and spread plated a LB+Kan agar plates and incubated at 370C overnight.
4.9 Isolation of plasmid from transformed cells
Colonies on LB+Kan plates were inoculated in LB broth and incubated overnight at 370C. Plasmid isolation of the cells was carried out using plasmid isolation kit MKT 04 of Chromous Boiotech, India. 1.5 ml of bacterial culture was taken in a MCT and centrifuged at 5,000 rpm for 5 mins. at room temperature. Supernatant was descarded and to the pellet added 150 µl of solution A (added with RNase A). Vortexed to break pellets completely. Added 150 µl of solution B and mixed by inverting MCT. Immediately added 150 µl of solution C and mixed gently by inverting. Centrifuged at 10,000 rpm for 15 mins at 40C. Transferred the clear supernatant in new MCT and discarded the pellet. Added 450 µl of precipitation solution and mixed by inverting the MCT. Centrifuged at 10,000 rpm for 15 mins. at room temperature. Decanted the supernatant and air dried the pellet for 10 mins. Suspended the pellet in 100 µl of 1X TE buffer by tapping. Isolated plasmid was subjected to 1% agarose gel electrophoresis along with 'blank' plasmid vector (without the murA insert).
5.0 Induced over-expression of murA
Earlier the transformation was carried out in DH5α strain of E.coli which has an improved transformation capability, now for production, the isolated plasmid was transformed into BL21 strain. Competent cell preparation, plasmid isolation and transformation steps remain the same.The resultant E.coli cells were inoculated in 200 ml LB+Kan broth and incubated overnight at 370C.Pipetted out 100 μl of the culture into clean MCTs and placed the tubes on ice until needed for gel analysis. To the rest of the culture in each tube added IPTG to a final concentrationof 1 mM. Incubated with shaking at 230 rpm at 37°C for 2 hours. Placed the cultures on ice. Pipetted 20 μl of each of the induced cultures into clean microcentrifugetubes. Added 20 μl of 2- SDS gel sample buffer to each microcentrifuge tube.Mixed the non-induced samples held on ice to resuspend the cells. Pipetted20 μl from each tube into a cleanmicrocentrifuge tube. Added 20 μl of 2-SDS gel sample buffer to each of the 20-μl aliquots of cells.Heated all tubes to 95°C for 5 minutes. Loaded the associated non-induced and induced samples in adjacent lanes for analysis by SDS-PAGE. Stain the protein gel with Coomassie Brilliant Blue stain.