Uv vis spectrophotometer

The UV-visible absorption spectrum of theaflavin monogallate (10 μM) was recorded using UV-vis Spectrophotometer (Amersham Bioscience, Sweden). The fluorescence emission of spectra of lactoferrin (5 μM) was recorded by exciting at 295 nm. All the fluorescence and absorbance readings were normalized to 1. The overlap between the fluorescence spectrum of lactoferrin and the absorption spectrum of the theaflavin monogallate was determined, and the FRET parameters were determined as reported previously.^{32 (link)}

Chlorophylls a, b and total carotenoids were determined using the procedure described by Teimouri et al.^{57 (link)}. Pigments were determined by adding 10 mL of acetone to 1 g of fresh tissue, followed by incubation at room temperature with agitation overnight. Samples were then centrifuged at 1500g for 5 min and measured using UV–Vis spectrophotometer (Amersham Biosciences, Little Chalfont, UK). The pigment content was calculated using the equations described by Hynstova et al.^{58 (link)}.

Primary cultures of E. coli expressing hGH and L-asparaginase were grown overnight in modified Luria–Bertani (LB) media (5 g/L glucose, 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl) from the glycerol stock. Antibiotics such as ampicillin (Amp) and kanamycin (Kan) were added into the primary culture of hGH, whereas only ampicillin was used in that of L-asparaginase. The final concentration of Kan and Amp used was 50 and 100 μg/ml, respectively. A medium containing suitable antibiotic(s) was inoculated with 1 ml glycerol stock of E. coli and incubated overnight at 37°C and 200 rpm in an orbital shaker (Kühner shaker, Switzerland).
Primary cultures of E. coli cells [1–2% (v/v) final concentration] grown overnight were inoculated into 1 L of LB media containing ampicillin (100 μg/ml) and kanamycin (50 μg/ml) antibiotics and were incubated at 37°C and 200 rpm. The growth of E. coli broth was monitored by measuring absorbance at 600 nm in a UV–Vis spectrophotometer (Amersham Biosciences, United Kingdom). At an OD of 0.6, the culture was induced by adding IPTG as an inducer wherein the final concentration of IPTG was 1 mM. The bacterial cultures were harvested 4 h postinduction by centrifugation at 4°C at 4,000 rpm for 15 min (Sorvall RC6+, United States) and stored at −20°C for further use.