Transformation of calcium-competent cells was carried out by the procedure detailed below: •The competent bacterial cells were thawed briefly and 200 μL of cells was mixed rapidly with plasmid DNA (10-50 ng) in fresh, sterile microcentrifuge tubes and maintained on ice for 30 min. A negative control with competent cells only (no added DNA) was also included. •Cell membranes were disrupted by subjecting cells to heat-pulse (42 °C) for 90 sec. •After heat shock, cells were incubated on ice for 5 min. •Cells were then mixed with 1 mL LB medium and incubated with shaking at 37 °C for 1 h. •For blue/white screening 40 μL of X-gal solution (20 mg mL-1 in dimethylformamide) and 4 μL of the IPTG (200 mg mL-1) was spread on LB-ampicillin (LB-amp) plates with a sterile glass rod. The plate was allowed to dry for 1h at 37 °C prior to spreading of bacterial cells. •Bacterial cells (100-200 μL) were spread and the plate was incubated at 37 °C for overnight. •White colonies were picked from the plates and suspended into LB-amp broth and cultivated to OD600=0.5

2 mL of an overnight culture of E. coli cells was inoculated into 100 mL LB medium and incubated with vigorous shaking at 30 °C until A600 of 0.8 was reached. •Cells were collected in 50 mL plastic (Falcon) tubes, cooled for 15 min on ice and centrifuged in a pre-cooled centrifuge (4,000 rpm for 10 min at 4 °C). •The pellet was suspended in 20 mL of ice-cold 50 mM CaCl2-15% glycerol solution, maintained on ice for 15 min and centrifuged again at 4,000 rpm for 10 min at 4 °C. •Pellet was resuspended in 2 mL of ice-cold 50 mM CaCl2-15 % glycerol solution, kept on ice for 30 min and aliquoted in 400 μL in microcentrifuge tubes. These were stored at -80 °C until required.

Preparation of electrocompetent cells (E. coli cells) A protocol was employed. The procedure was carried out in cold under sterile conditions as follows: •A single colony of E. coli DH10B/ DH5α/XL1blue was inoculated in 20 mL of LB medium and grown overnight at 30 °C. •500 mL LB medium was inoculated with 5mL of this overnight grown culture of the E. coli and incubated with vigorous shaking (250 rpm) at 30 °C until an A600of 0.5 - 0.8 was achieved. •The cells were chilled in ice for 10-15 min and transferred to prechilled Sorvall® centrifuge tubes and sedimented at 4,000 rpm for 20 min at 4 °C. •The supernatant was decanted and cells were resuspended in 500 mL of sterile ice-cold water, mixed well and centrifuged as described above. •The washing of the cells described above was repeated with 250 mL of sterile ice-cold water, following which cells were washed with 40 mL of ice-cold 10 % (v/v) glycerol and centrifuged at 4,000 rpm for 10 min. •The glycerol solution was decanted and the cell volume was recorded. The cells were resuspended in an equal volume of ice-cold 10 % glycerol. •Cells were then dispensed in 40 μL volumes and stored at -80 °C until required.

In order to minimize self ligation of vector during cloning experiments, the digested DNA was subsequently treated with calf intestinal phosphatase (CIP) [NEB, UK]. The reaction conditions and amount of CIP were optimized and varied from (0.06-1) unit/picomole DNA termini. The dephosphorylation reaction was carried out in 50 μL reaction as follows. Reaction mixture containing no restriction enzyme was treated as control. Reaction was incubated for 1 h at 37 °C and stopped by heat inactivation at 65 °C for 20 min. 2.5.5. Composition of restriction mixture (50 μL) Linearized Plasmid DNA X μL (1 μg) CIP 1 μL (0.06-1 U μL-1) Reaction buffer (10X) 5.0 μL Distilled water Y μL Total volume 50 μL Linearized and dephosphorylated plasmids from each reaction were purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany). 100 ng DNA from each reaction was then ligated in15 μL reaction volume containing 1.5 μL of 10X ligation buffer (NEB, England) and 0.2 μL of T4 DNA ligase to check the efficiency of self ligation after dephosphoryaltion. The ligation mixture was incubated at 16 °C for overnight and transformed into E. coli DH5αcompetent cells.

cryopreserved culture vial was obtained from the liquid nitrogen tank, and thawed quickly at 37°C in a water bath. To the vial, O.lv of 12% NaCl was addeq I slowly, dropwise, while shaking the tube gently. Subsequently, 10v of 1.6% NaCl I was added slowly, dropwise while swirling the tube, followed by centrifugation at 200 g at 20°C for 5 min. The supernatant was discarded and 10v of RPMI 1640 complete media was added, followed by centrifugation at 200 g at 20°C for 5 min.' After removal of the supernatant, pelleted parasites were resuspended in complete I media at 0.5% hematocrit. Cultures were gassed with 5% C02, 3% 02, and 92%' N2 and maintained at 37°C

Revival of cryo-preserved Plasmodiumfalciparum cultures

DNA restriction enzymes were purchased from New England Biolabs (Massachusetts, USA) and Life Technologies (Maryland, USA). Lysozyme and RNase A were obtained from Sigma. RNase Tl, DNA ligase, RNA polymerase, Taq DNA polymerase, lKb DNA ladder and prestained molecular weight markers for· proteins were obtained from Life Technologies (Maryland, USA). Other protein molecular weight markers were from Sigma chemical co. T4 polynucleotide kinase were purchased from Promega. T7 DNA polymerase was obtained from USB.

Enzymes and Molecular Weight Markers

Restriction endonucleases, T 4 DNA 1 igase, DNA polymerase I large fragment Klenow ) , bacterial alkaline phosphatase BAP were from BRL, USA and New England Biolabs, USA. Lysozyme and RNase A were from Sigma. Thermus aquaticus thermostable DNA polymerase was kindly provided by Cetus Corporation, California, USA.

Enzymes.

32P-a-rUTP (3000Ci/mmol) was obtained from Perkin Elmer (California, USA).

Radioisotopes

Nutrient agar (NA) medium The composition per litre of the medium is as follows: Peptone : 5.00 g Sodium chloride : 8.00 g Beef extract : 1.50 g Yeast extract : 1.50 g Agar-agar : 20.0 g Double distilled water : to make the final volume 1000 ml (iii) Tannic acid agar (TAA) medium This medium was used for screening of tannase producers. The composition per litre of the medium is as follows: Tannic acid : 10.00 g Agar-agar : 30.00 g Citrate phosphate buffer : to make the final volume 1000 ml (0.1M, pH 5.0) 30.0 g of agar-agar was melted and subsequently autoclaved. Citrate phosphate buffer and 0.1% (w/v)tannic acid, filter sterilized through 0.22μmembrane filters, were added to the sterilized molten agar and the final volume was made 1.0 L. (IV) Czapek Dox minimal medium (modified for tannase production)The composition per litre of the medium is as follows: Ingredients Fungi Bacteria Tannic acid : 10.00 g 10.00 g D-Glucose : 10.00 g 0.50 g NaNO3 : 6.00 g – NH4Cl : – 1.0 g KH2PO4 : 1.52 g 0.50 g K2HPO4 : – 0.50 g KCl : 0.52 g – MgSO4.7H2O : 0.52 g 0.50 g CaCl2 : – 0.01 g Cu(NO3)2.3H2O : trace – FeSO4.7H2O : trace – ZnSO4.7H2O : trace – Double Distilled water : to make 1.0 L to make 1.0 L pH : 5.0±0.2 5.0±0.

Potato dextrose agar (PDA) medium The composition per litre of the medium is as follows: Ingredients g/l Peeled and sliced potatoes : 200.0 Dextrose : 20.0 Agar-agar : 20.0 Double Distilled water : to make the final volume 1000 ml pH was adjusted to 6.2 ± 0.2 using 1N NaOH / HCl

Medium Compositio

Z broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

Z broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

LB mediumTryptone 10.0 g Yeast Extract 5.0 g NaCl 10.0 g H2O to 1000 ml pH was adjusted to 7.0 –7.2 with 1 N NaOH.LB agar LB medium 1000 ml Bacto-agar 15.0g LB soft agar LB medium 100 ml Bacto-agar 0.6 g

H2O to 1000 ml LBON medium (LB medium without NaCl) Tryptone 10.0 g Yeast Extract 5.0g H2O to 1000 ml pH was adjusted to 7.0-7.2 with 1N NaOH. LBON agar LBON medium 1000 ml Bacto-agar 15.0gMacConkey Agar MacConkey agar (Difco) 51.5 g H2O to 1000 ml

LB mediumTryptone 10.0 g Yeast Extract 5.0 g NaCl 10.0 g H2O to 1000 ml pH was adjusted to 7.0 –7.2 with 1 N NaOH.LB agar LB medium 1000 ml Bacto-agar 15.0g LB soft agar LB medium 100 ml Bacto-agar 0.6 gZ broth (for P1 transduction) LB medium 100 ml 0.5 M CaCl20.5 ml Buffered LBagarTryptone 10.0 gYeast extract 5.0 gMin A salts1XBacto-agar15.0 gH2O to 1000 ml Buffered Yeast extract agarYeastExtract 5.0 gMin A salts 1X Bacto-agar15.0 gH2O to 1000 ml Yeast extract brothYeast Extract 5.0 g NaCl 10.0 g Bacto-agar15.0 g

All media and buffers were sterilized by autoclaving at 121ºC for 15 minutes. Media and buffers used in this study are given below: Glucose Minimal A medium Minimal A salts(1X)K2HPO410.5g KH2PO44.5g (NH4)2SO41.0g CH3COONa.2H2O 0.5g H20 to 1000 ml After autoclaving the following solutions were addedto Min A salts: MgSO4(1M)1 ml Glucose (20%) 10 ml Vitamin B1 (1%) 0.1 ml Amino acids when required were added to a final concentration of 40 μg/ml or casaminoacids were added at a concentration of 0.2% whenever needed.Minimal A agar It contains 1.5% bacto-agar (Difco) in Minimal A medium. The plates were poured after mixing double strength Minimal A with 3% agar.M9 minimal medium(1X)Na2HPO4•7H2O7.0 gKH2PO43.0 gNaCl0.5gNH4Cl1.0 gH20to 1000 mlSterilize the solution by autoclaving.Glucose-M9 minimal medium was madein asimilarwayto that of Glucose Minimal A medium

Media