OFAT method was carried out by changing parameters of factors in a sequential manner. The factors considered for optimization were pH of the media, Incubation temperature, Carbon and nitrogen sources and presence of metal ions. The pH of the media was adjusted between 4 and 10. The incubation temperature ranged between 4 °C and 45 °C. Additional carbon sources like glucose, fructose, sucrose, dextrose, inositol, mannitol, rhamnose, maltose, lactose, trehalose, sodium citrate, arabinose (HiMedia Laboratories, Mumbai, India, LR grade) was supplemented at 10 g/L along with Starch. The effect of nitrogen source on amylase production was evaluated by replacing peptone with malt extract, beef extract, yeast extract, peptone, casitone, corn steep liquor (HiMedia Laboratories, Mumbai, India, LR grade) and inorganic nitrogen sources such as ammonium sulphate, ammonium chloride, ammonium carbonate, potassium nitrate and sodium nitrate (HiMedia Laboratories, Mumbai, India, LR grade). Various metal ions such as Fe2+, Cu2+, Mg2+, K+, Ca2+, Mn2+, Ba2+, Sn2+, Zn2+, Co2+, Ni2+at 0.5 g/L were supplemented in the media in the form of metal salts. All the experiments were performed in triplicates and the data is represented in Mean ± Standard deviation.
Rhamnose
Manufactured by HiMedia
Sourced in India
Rhamnose is a monosaccharide that functions as a building block for various biomolecules. It is a C6 sugar with the chemical formula C₆H₁₂O₅.
Automatically generated - may contain errors
Lab products found in correlation
Isopropyl β d 1 thiogalactopyranoside (iptg), by HiMedia (2 mentions)
Superdex 200 column, by GE Healthcare (2 mentions)
Luria bertani broth, by HiMedia (2 mentions)
Dextrose, by HiMedia (1 mentions)
Maltose, by HiMedia (1 mentions)
Trehalose, by HiMedia (1 mentions)
Ammonium sulphate, by HiMedia (1 mentions)
Arabinose, by HiMedia (1 mentions)
Mannitol, by HiMedia (1 mentions)
Peptone, by HiMedia (1 mentions)
Sucrose, by HiMedia (1 mentions)
Fructose, by HiMedia (1 mentions)
Lactose, by HiMedia (1 mentions)
Yeast extract, by HiMedia (1 mentions)
Malt extract, by HiMedia (1 mentions)
Beef extract, by HiMedia (1 mentions)
4 protocols using rhamnose
1
Optimization of Amylase Production
2
Purification of Cdh23 EC Repeats
3
Cadherin-23 Extracellular Repeat Expression
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Mouse Cdh23 (NP_075859.2) extracellular (EC) repeats EC1 (Q24 to D124) and EC1‐2 (Q24 to D228) were PCR‐amplified and cloned into pET21a vector (Novagen, Merck‐Sigma, St. Louis, MO, USA). We considered the Q24 as Q2 in our experiments. Cdh23‐EC1, EC1‐2, and EC1‐3 repeats were expressed in E. coli BL21CodonPlus (DE3)‐RIPL cells (Stratagene, San Diego, CA, USA) and E. coli lemo(DE3) cultured in Luria/Bertani broth (Hi‐Media, Mumbai, Maharashtra, India) and grown at 37 °C to an OD600 of 0.6. Induction was carried out differently for both the strains. For expression in E. coli lemo(DE3), the cells were induced along with 1 mm rhamnose and 0.2 mm IPTG (Hi‐Media) and then induced at 30 °C for 6 h. For expression in E. coli BL21CodonPlus (DE3)‐RIPL, the protein was then induced with 0.2 mm IPTG and incubated at the same temperature. Unlike in E. coli BL21CodonPlus (DE3)‐RIPL, Cdh23 EC1‐2 protein from E. coli lemo(DE3) was obtained in the soluble fraction. For expressions in E. coli BL21CodonPlus (DE3)‐RIPL, we revived the proteins from the inclusion bodies and processed through serial dialysis, as reported 8 . All proteins were first purified using Ni‐NTA beads followed by SEC on a Superdex 200 column (GE Healthcare, Chicago, IL, USA) in 25 mm HEPES (pH 7.5), 25 mm KCl, 100 mm NaCl, and 2 mm CaCl2.
4
Rhamnolipid Production Assay in P. aeruginosa
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The effect of EPIs on rhamnolipids was assessed by following a method described earlier [65 (link)], with some modifications. The OD600nm of P. aeruginosa PAO1 and PAO750 was adjusted to 0.5, diluted 10 times in fresh PPGAS (0.02 M NH4Cl2, 0.02 M KCl, 0.12 M Tris-HCl, 1.6 mM MgSO4, 0.5% glucose, 1% peptone, pH 7.2) [66 (link)] medium, and incubated at 37°C for 24 h in the presence of compounds (Ar1, Ar5, Ar11, and Ar18) at 16 μg/mL. After 24 h, the culture was centrifuged at 13,000 ×g for 15 min. Then, 500 μL of supernatant was added to 1 mL of diethyl ether, shaken, and let it settle down. Next, 900 μL of the upper organic layer was collected and subject to dry at 80° C for 15 min. Afterward, 900 μL of orcinol-reagent (0.19% orcinol in 53% sulphuric acid) was added, incubated at 80°C for 30 min, cooled at room temperature (for 15 min), and OD421nm was measured. The amount of rhamnolipids in samples was generated using a standard curve of OD421nm versus concentration of rhamnose (Himedia, India).
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