Ethyl alcohol, ammonia, diethyl ether, hexane, acetonitrile, isopropyl alcohol, anhydrous sodium sulfate, and squalene standard were obtained from Sigma Aldrich (St. Louis, MO, USA).
Squalene standard
Manufactured by Merck Group
Sourced in United States, India
Squalene standard is a pure, high-quality reference compound used for analytical purposes. It serves as a calibration standard to quantify and identify squalene in various samples through analytical techniques such as chromatography and spectroscopy.
Automatically generated - may contain errors
Lab products found in correlation
Acetonitrile, by Merck Group (2 mentions)
Hexane, by Merck Group (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Anhydrous sodium sulfate, by Merck Group (1 mentions)
Ethyl alcohol, by Merck Group (1 mentions)
Isopropyl alcohol, by Merck Group (1 mentions)
Ptfe syringe filter, by Avantor (1 mentions)
Heptane, by Merck Group (1 mentions)
Enzyme, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
D xylose, by Merck Group (1 mentions)
2 propanol, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Potassium hydroxide, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Span 80, by Merck Group (1 mentions)
7 protocols using squalene standard
1
Extraction and Quantification of Squalene
2
Squalene Extraction from Cell Cultures
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Cells were harvested from 10-20 ml of culture and squalene was extracted according to a protocol adapted from Schmerk et al (2011) (link) (Schmerk et al., 2011 (link); Englund et al., 2015 (link)). Pellets were resuspended in 5ml of 10% KOH (Merck) in methanol (w/v) (Merck) and vortexed repeatedly until completely suspended. The samples were then incubated at 65 °C for 1 h, followed by addition of 2 ml of heptane (Sigma-Aldrich) and mixing. The upper phase was transferred to a new tube and dried at room temperature under a stream of N2 before being redissolved in heptane and acetonitrile to a final ratio of 1:20 v/v. The solution was filtered through 0.2 mm PTFE syringe filters (VWR International, Radnor, PA, USA), and the squalene content was determined by HPLC with comparison to a squalene standard (Sigma-Aldrich) (Englund et al., 2014 ).
3
Biosynthesis of Squalene and Carotenoids
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Squalene standard
(≥98%), FPP (≥95%), and GGPP (≥95%) were purchased
from Sigma (Sigma-Aldrich, USA). Methanol, acetone, ethyl acetate,
acetonitrile,d -xylose, and 2-propanol were purchased from
Merck (Merck, Germany). Enzymes were obtained from Thermo (Thermo
Fisher Scientific, USA). Other culture media components were purchased
from Duchefa (Duchefa Biochemie, The Netherlands).
Escherichia coli DH5α and B.
subtilis 168 (Novagen, The Netherlands) were used
for gene cloning and protein expression, respectively. CrtM and crtN genes were obtained from S. aureus. The SQS gene was obtained
from Bacillus megaterium (GenBank:ADF40697.1 ).
All the plasmids and strains constructed in this study are listed
inTable S1 . The primers (Table S2 ) used in this study were synthesized by Eurofins
(Eurofins Scientific, The Netherlands).
(≥98%), FPP (≥95%), and GGPP (≥95%) were purchased
from Sigma (Sigma-Aldrich, USA). Methanol, acetone, ethyl acetate,
acetonitrile,
Merck (Merck, Germany). Enzymes were obtained from Thermo (Thermo
Fisher Scientific, USA). Other culture media components were purchased
from Duchefa (Duchefa Biochemie, The Netherlands).
Escherichia coli DH5α and B.
subtilis 168 (Novagen, The Netherlands) were used
for gene cloning and protein expression, respectively. CrtM and crtN genes were obtained from S. aureus. The SQS gene was obtained
from Bacillus megaterium (GenBank:
All the plasmids and strains constructed in this study are listed
in
(Eurofins Scientific, The Netherlands).
4
Quantitative Analysis of Squalene and Ergosterol
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) were procured from Merck chemicals, (Bangalore, India). Cyclohexane has been used as a solvent system for squalene analysis while petroleum ether: diethyl ether: acetic acid in the ratio 90:10:1 has been used as a solvent system for ergosterol analysis. Once the mobile front reached the desired height on the silica, the plates were removed from glass chamber and were exposed to 20% H 2 SO 4 mist and later incubated for heating at 70°C in hot air oven for visualization. The bands were also visualized in UV chamber . CC-BY-NC-ND 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was not this version posted April 28, 2018. ; https://doi.org/10.1101/309609 doi: bioRxiv preprint as the squalene has an absorbance of 195 nm. The spot intensity was compared with the squalene standard obtained from Sigma-Aldrich (Bangalore, India).
The copyright holder for this preprint (which was not this version posted April 28, 2018. ; https://doi.org/10.1101/309609 doi: bioRxiv preprint as the squalene has an absorbance of 195 nm. The spot intensity was compared with the squalene standard obtained from Sigma-Aldrich (Bangalore, India).
5
Yeast and E. coli Strains for Lipid Biosynthesis
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Euroscarf yeast strains BY4742, CEN.PK2-1C, ERG11Δ, ERG6Δ and E.coli strain DH5α were kindly provided by Prof. Ram Rajasekharan (Lipid Science Dept., CSIR-CFTRI). Squalene standard was procured from Sigma-Aldrich (Bangalore, India). The restriction enzymes, ligase and phusion DNA polymerases were obtained from New England Biolabs, (Ipswich, MA, USA).
Yeast strains BY4742, CEN.PK2-1C, ERG11Δ, ERG6Δ and recombinant strains were grown at 30°C either in YPD or synthetic medium without uracil(Paramasivan and Mutturi 2017b). E. coli DH5α was grown at 37°C in LB Media (10 g tryptone/L, 10 g yeast extract/L and 5 g sodium chloride/L) in the presence or absence of ampicillin (100 µg/mL) as per requirement and is used for the propagation of plasmids. Cell growth was determined by measuring the OD 600 .
Yeast strains BY4742, CEN.PK2-1C, ERG11Δ, ERG6Δ and recombinant strains were grown at 30°C either in YPD or synthetic medium without uracil(Paramasivan and Mutturi 2017b). E. coli DH5α was grown at 37°C in LB Media (10 g tryptone/L, 10 g yeast extract/L and 5 g sodium chloride/L) in the presence or absence of ampicillin (100 µg/mL) as per requirement and is used for the propagation of plasmids. Cell growth was determined by measuring the OD 600 .
6
Isolation and Characterization of Mangrove-Associated Microbes
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All carbon and nitrogen sources were obtained from Merck (Darmstadt, Germany). Standard squalene was obtained from Sigma (St. Louis, Mo., U.S.A.). All other chemicals and solvents were provided in analytical grade. Seawater samples, fallen leaves of mangrove trees (Avicennia marina and Rhizophora mucronata) and sediments were collected from coastal waters and mangrove forest of Persian Gulf and Oman Sea in south of Iran (26°50′36.0"N 55°41′13.2"E and 26°19′30.9"N 57°07′28.0"E). Samples were kept in sterile bags at 4 °C and sent to the laboratory before use. Also, natural seawater sample with chemical composition including 7810 mg/L Na+, 22,961 mg/L Cl−, 410 mg/L Ca2+, 1386 mg/L Mg2+, 259 mg/L K+ and 2479 mg/L SO42− was used for preparation of culture media.
7
Pumpkin Seed Oil Extraction and Analysis
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Potassium hydroxide (KOH), FeCl3⋅6H2O (97%), FeCl2⋅4H2O (99%), acetone, ethanol, hexane, ammonium hydroxide (27–30%), Tween 80, and Span 80 were purchased from Merck Company (Darmstadt Merck, Germany). Pumpkin seed oil was procured from Zarband Company, Iran. Standard squalene was purchased from Sigma Aldrich Company for Gas Chromatography (GC) analysis.
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