Flowchart 5.1. Summary of Chapter 5
7.2 Materials and Methods
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also found to inhibit appresoria formation and conidial germination of fungi. The effectiveness in the dual exploitation of glufosinate ammonium along with the fungi makes it a lucrative option in transformation as it can be used as a dominant selectable marker as well as the recombinant fungus can be used to control pests in combination with this herbicide.
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and inoculated to 5 ml of liquid LB media and cells were allowed to grow overnight at 37 °C at 200 rpm. A 200 µl of the culture was inoculated in 50 ml of LB media and incubated at 37 °C and 200 rpm until the OD590 reaches 0.7. Cells were kept on ice for 20 min and centrifuged at 5000 rpm at 4 °C for 10 min. Cell pellet was gently resuspended in 1/10th volume of TSS buffer (LB medium 85 % (v/v), PEG 10 % (w/v), Dimethyl sulfoxide 5 % (v/v), Magnesium chloride 50 mM) and incubated on ice for 20 min. A 200 µl of cells were aliquoted in prechilled microcentrifuge tubes and stored immediately at -20 °C until further use.
Transformation of plasmid pBARGEM7-2 to E. coliDH5α
The transformation mixture contained 4 µl of plasmid DNA (pBARGEM7-2) & 100 µl of fresh competent cells. The mixture was incubated on ice for 30 min and after the incubation immediately subjected to heat shock at 42 °C for 60 sec. A 900 µl of pre warmed (37- 42 °C) SOC medium was added to it. Thereafter the mixture was again incubated on ice for 5 min. The mixture was incubated for 1 h at 37 °C and 200 rpm. The transformed colonies were grown and selected in LB agar media containing ampicillin (100 µg/ ml).
Minipreparation of plasmid DNA
Plasmid (pBARGEM7-2) was extracted from transformed E. coli DH5α cells by minipreparation of plasmid DNA (Fig 7.1b). A single colony of transformed E. coli DH5α was inoculated in 5 ml of liquid LB media supplemented with 50 µg/ ml ampicillin and incubated overnight at 37 °C and 200 rpm. A 2 ml of that culture were taken in a microcentrifuge tube and spinned at 10000 rpm for 1 min, supernatant was discarded. The pellet was suspended in 100 µl of ice cold solution 1 [50 mM glucose, 25 mM Tris HCl (pH 8.0), 10 mM EDTA (pH 8.0)] and
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incubated for 5 min. A 200 µl of freshly prepared solution 2 [0.2 N NaOH, 1 % (w/v) SDS] was added and mixed thoroughly by inverting the tubes and incubated for 3 min on ice. Thereafter solution 3 [60 ml of 5 M potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml Millipore water]
was added and mixed gently and incubated for 5 min on ice. The mixture was centrifuged at 10000 rpm for 15 min at 4 °C and supernatant was transferred to fresh centrifuge tube. A 450 µl of isopropanol was added and tube was kept at -20 °C. The quality of the plasmid DNA was checked on 1 % agarose gel.
Transformation of plasmid pBARGEM7-2 to fungal protoplasts
A modified method of Herrera-Estrella et al. (1990) was adapted for transformation experiments. Transformation mixture consisted of 200-300 µl protoplast solution (6×107 protoplasts/ ml) in 10 ml centrifuge tube along with 15 µg of plasmid pBARGEM7-2. Poly ethylene glycol (PEG 8000; Sigma), 25 % (w/v), in 10 mM Tris-HCL (pH 7.5) containing 50 mM CaCl2 was added and tubes were incubated for 30 min on ice. A 2 ml of PEG solution was added again and mixed gently. After 5 min of incubation at room temperature the solution was diluted by adding 4 ml 0.7 M KCL and 50 mM CaCl2. A 200 µl of diluted solution is plated on SDA containing 200 µg/ ml of glufosinate ammonium as minimum inhibitory concentration of glufosinate ammonium was determined to be 200 µg/ ml.
Selection and Stabilization of Transformants
Stable transformants were selected after repeated sub-culturing for four generations by transferring colonies to glufosinate ammonium containing SDA plates and allowing the fungus to grow until sporulation.
196 Extraction of DNA
Mycelium was harvested from four day old SD broth by centrifugation at 8000 rpm for 15 min and dried using blotting paper. Mycelium was transferred to a sterile porcelain mortar and crushed with liquid nitrogen. 10 mg of this crushed mycelial powder was taken into a microcentrifuge tube and 500 µl of lysis buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA, 3 % SDS) were added. After incubation at 65 °C for 1 h, 500 µl of TE saturated phenol/ chloroform (1:1) was added and after vigorous vortexing the mixture was centrifuged at 13000 rpm for 20 min. This step was repeated for several times to remove all proteins. Supernatant transferred to a new microcentrifuge tube and 0.1 volumes of 3 M Na acetate and 1 volume of ice cold isopropanol added. The solution allowed to stand on ice for 2 h and centrifuged at 13000 rpm for 20 min. DNA pellet was washed with 70 % ethanol, air dried and dissolved in Tris EDTA buffer.
RNA was removed by incubating the DNA with 2 µl of 10 mM RNAse A for 30 min at 37 °C.
Thereafter the phenol/ chloroform extraction was repeated and DNA pellet was recovered with Na acetate and isopropanol. After washing with 70 % ethanol, air dried and dissolved in Tris EDTA buffer and stored at -20 ºC in aliquots.
Confirmation of the transformation
DNA was isolated by the above described method. Molecular confirmation was achieved by a confirmation PCR using Primers, specifically designed for bar gene based on the conserved domains of the bar gene product.
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Forward primer GGCGGGCTATGCGTATGCGA
Reverse primer GCAATCACCGCAATCATCTGG
In order to achieve biochemical confirmation, native & transformed isolates were grown in Czapek-Dox broth supplemented with glufosinate ammonium (100 µg/ ml) and the culture supernatants were analyzed by SDS-PAGE
Features of the original and transformed isolate
Virulence determinant enzymes, Chitinase and Protease; percentage of germination along with sporulation was studied for both the Native isolates and Transformed ones.