. Jalciparum cultures were maintained as described previously (Trager and Jensen, 1976). Briefly, P. Jalciparum strain 3D7 was cultured at 37°C in RPMI " 1640 medium (list I) in 0+ RBCs supplemented with 10% AB+ human serum or : 0.5% Albumax II (complete medium). All media were preheated to 37°C and care was taken to minimize the handling time outside the 37°C incubator. Cultures were gassed with 5% CO2, 3% O2, and 92% N2 for 20 seconds and maintained at 37°C.

Maintenance of P.falciparum cultures

Oligonucleotides used in this study were synthesized by Rama Biotechnologies (Hyderabad, India).

Oligonucleotides

Ampicillin, tetracycline, chloramphenicol and gentamycin were from Sigma. Geniticin ( G418 ) was from Gibco Laboratories, USA.

Antibiotics.

DNA restriction enzymes were purchased from New England Biolabs (Massachusetts, USA) and Promega Corporation, (Madison, U.S.A.). RNase A was obtained from Qiagen (West Sussex, U.K.). DNA ligase, RNA polymerase, RNAsin, Taq DNA polymerase, T7 RNA polymerase, SP6 RNA Polymerase and alkaline phosphatase were obtained from Promega Biotech.

Enzymes

For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity

For fungal cultures, spores were harvested from 72 hour old cultures grown on PDA/Tannic acid agar slants by adding 10 ml of sterilized normal saline and a few drops of sterilized Tween-80 followed by vortexing. The spore suspension was filtered through sterile cotton filter to ensure that mycelial filaments are removed. The spores were counted using a haemocytometer (Neubaeur). Approximately, 5X106 spores were inoculated in 50 ml of tannase production medium in 250 ml Erlenmeyer flasks. These flasks were then incubated at 30±1 and 37±1°C in an incubator shaker (model G25KC, New Brunswick Scientific, NJ, USA) at 200 rpm

EDTA 2 mMThis was prepared at 50X concentration and used at 1X concentration. Both TBE and TAE were used as standard electrophoresis buffers.Gel loading buffer with dye Tris-Cl (pH 7.5) 250 mM Bromophenol blue/ Xylene cyanol 0.02% Glycerol 20%INOUE (PIPES) Buffer PIPES (Free acid) 10 mM CaCl2.2H2O 15 mM KCl 250 mM MnCl2.4H2O 55 mM pHwas adjusted to 6.7 with 1N KOH. PIPES gets into solution when the pH is greater than 6.7. MnCl2 was dissolved separately and added with stirring. The pH was then adjusted to 6.7 and solution wasfilter sterilized and stored at –20ºC.Z Buffer (for β-galactosidase assay) Na2HPO416.1 g NaH2PO45.5 g KCl 0.75 gMgSO4.7H2O 0.246 g H2O to 1000 ml pH was adjusted to 7.0 and stored at 4ºC.Pre-Hybridization Buffer 20X Saline-sodium citrate (SSC)3ml50% dextran sulphate2ml50X Denhardt’s solution1m

20%SDS250 μl10 mg/ml Salmon sperm DNA100 μlDEPC waterto 10mlHybridization Buffer Same as pre-hybridisation buffer but contains the radio-labelled probe.SDS sampleBuffer(1X)Tris-HCl, pH 6.850mMGlycerol10%EDTA12.5 mM SDS2%Bromophenol blue0.02%β-mercaptoethanol1%Running buffer for western blottingGlycine14.4 g/lTris base3.05 g/lSDS1.0 g/lTransfer buffer for western blottingGlycine14.4 g/lTris base3.03 g/lThe above salts were dissolved in 800 ml of milliQ water and 200 ml of methanol was then added. The buffer was chilled before use.TBST buffer for Western blot10X of TBS (1000ml)Sodium chloride80 gPotassium chloride2 gDisodium hydrogen phosphate(Na2HPO4)14.1 gPotassium dihydrogen phosphate(KH2PO4)2.49 gMilliQ waterto 1000m

EDTA 2 mMThis was prepared at 50X concentration and used at 1X concentration. Both TBE and TAE were used as standard electrophoresis buffers.Gel loading buffer with dye Tris-Cl (pH 7.5) 250 mM Bromophenol blue/ Xylene cyanol 0.02% Glycerol 20%INOUE (PIPES) Buffer PIPES (Free acid) 10 mM CaCl2.2H2O 15 mM KCl 250 mM MnCl2.4H2O 55 mM pHwas adjusted to 6.7 with 1N KOH. PIPES gets into solution when the pH is greater than 6.7. MnCl2 was dissolved separately and added with stirring. The pH was then adjusted to 6.7 and solution wasfilter sterilized and stored at –20ºC.Z Buffer (for β-galactosidase assay) Na2HPO416.1 g NaH2PO45.5 g KCl 0.75 gMgSO4.7H2O 0.246 g H2O to 1000 ml pH was adjusted to 7.0 and stored at 4ºC.Pre-Hybridization Buffer 20X Saline-sodium citrate (SSC)3ml50% dextran sulphate2ml50X Denhardt’s solution1ml

20%SDS250 μl10 mg/ml Salmon sperm DNA100 μlDEPC waterto 10mlHybridization Buffer Same as pre-hybridisation buffer but contains the radio-labelled probe.SDS sampleBuffer(1X)Tris-HCl, pH 6.850mMGlycerol10%EDTA12.5 mM SDS2%Bromophenol blue0.02%β-mercaptoethanol1%Running buffer for western blottingGlycine14.4 g/lTris base3.05 g/lSDS1.0 g/lTransfer buffer for western blottingGlycine14.4 g/lTris base3.03 g/lThe above salts were dissolved in 800 ml of milliQ water and 200 ml of methanol was then added. The buffer was chilled before use.TBST buffer for Western blot10X of TBS (1000ml)Sodium chloride80 gPotassium chloride2 gDisodium hydrogen phosphate(Na2HPO4)14.1 gPotassium dihydrogen phosphate(KH2PO4)2.49 gMilliQ waterto 1000ml

1litreof 1X TBS +1 ml of Tween-2040% Acrylamide solution (29:1) Acrylamide39 g Bis-acrylamide 1 g H2O to 100 ml7.5M Urea 10%acrylamidecomposition 40% Acrylamide 12.5mlUrea22.5g5X TBE 10ml DEPC treated H2O to 50ml The gel mixtures were filtered through a 0.45 μ Millipore filter before adding APS and TEMED

Citrate Buffer Citric Acid (0.1 M) 4.7 volumesSodium citrate (0.1 M)15.4 volumesTE Buffer Tris-Cl (pH 8.0) 10 mM EDTA 1 mMTBE Buffer Tris-Borate 90 mM EDTA 2 mMThis was prepared as 5X solution and used at 0.5X concentration.TAE Buffer Tris-Acetate 40 mM

Buffers and solutions