RU2499042C1 - METHOD FOR ISOTOPE ENRICHMENT OF E.coli CELLS - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method for enrichment of E.coli cells with magnesium isotopes provides for cultivation of E.coli cells during 10-16 h at the temperature of 37°C in water solution enriched with magnesium isotope ²⁴Mg or ²⁵Mg or ²⁶Mg. Water solution includes NH₄Cl, glucose, Na₂HPO₄, KH₂PO₄, NaCl and MgSO₄ at the following component content per litre of distilled water: NH₄Cl - 2 g, MgSO₄ - 200-260 mg, glucose (dry) - 8-10 g, Na₂HPO₄ - 11.9-12.1 g, KH₂PO₄ - 5.95-6.05 g, and NaCl - 0.98-1.02 g.

EFFECT: effective substitution of natural intracellular magnesium of Ecoli cells for the corresponding isotope.

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Description

The method of isotope enrichment of E. coli cells relates to microbiology, enzymology for the production of isotope-labeled cells of microorganisms.

The known method of isotope enrichment (RF patent No. 2399409, publ. 09/20/10), containing the stage of isotope exchange between an aqueous solution containing at least two components, each of which is represented by the formula H ₂ OH ₂ SiF ₆ · nSiF ₄ ( where n≥0), and with a gas containing SiF ₄ to enrich the stable Si isotope. It is possible that SiF _{4 is} dissolved in an aqueous solution to a saturation state, and also that the aqueous solution has an azeotropic composition.

The disadvantage of this method is the impossibility of its use for enrichment of E. coli cells, because This solution does not contain nutrients for bacteria and does not have a pH = 6.5-7.5, at which cells grow and multiply.

The technical result of the proposed method for the isotopic enrichment of E. coli cells is the effective replacement of natural intracellular magnesium with the corresponding isotope.

The problem is solved in that in the method of isotope enrichment of E. coli cells containing an isotope exchange step between E. coli cells and an aqueous solution, characterized in that the isotope exchange between E. coli cells and an aqueous solution is carried out for 10-16 hours at a temperature 37 ° C, wherein the aqueous solution enriched on one of magnesium isotopes ²⁴ Mg, ²⁵ Mg, ²⁶ Mg, includes NH ₄ Cl, dextrose, Na ₂ HPO _4, KH ₂ PO _4, NaCl and MgSO _4, at the following content in 1 liter of distilled water:

NH ₄ Cl - 2 g;

MgSO ₄ - 200-260 mg;

glucose (dry) - 8-10 g;

Na ₂ HPO ₄ - 11.9-12.1 g;

KH ₂ PO ₄ - 5.95-6.05 g;

NaCl - 0.98-1.02 g.

The method of isotopic enrichment of E. coli cells was carried out as follows.

The museum strain of Escherichia coli K12TG1 was previously incubated in Lb broth (manufactured by Sigma Aldrich Co.), which contains natural magnesium, for 24 hours at 37 ° C. Then, three microorganisms of initial concentration 10 ⁵ KOE / ml were sown in a synthetic nutrient medium M9 - an aqueous solution with a different concentration of 6 components per 1 liter of distilled water.

The first aqueous solution:

NH ₄ Cl - 2 g; MgSO ₄ - 200 mg; glucose (dry) - 8 g; Na ₂ HPO ₄ - 11.9 g; KH ₂ PO ₄ - 5.95 g; NaCl - 0.98 g.

The second aqueous solution:

NH ₄ Cl - 2 g; MgSO ₄ - 230 mg; glucose (dry) - 9 g; Na ₂ HPO ₄ - 12 g; KH ₂ PO ₄ - 6 g; NaCl - 1 g.

Third aqueous solution:

NH ₄ Cl - 2 g; MgSO ₄ - 260 mg; glucose (dry) - 10 g; Na ₂ HPO ₄ - 12, 1 g; KH ₂ PO ₄ - 6.05 g; NaCl - 1.02 g.

The first three substances, NH ₄ Cl, glucose (dry) and MgSO ₄ , enter the nutrient solution necessary to maintain the viability of the cell culture and its growth, required for the accumulation of isotope-enriched magnesium cell biomass. The last three substances, Na ₂ HPO ₄ , KH ₂ PO ₄ and NaCl, are a buffer solution that maintains pH = 6.5-7.5 medium. Two solutions are prepared separately, sterilized in an autoclave for 25 minutes at a pressure of 1.5-2 atm. After autoclaving, the solutions merge; pH control before and after sterilization. Magnesium isotopes are added to the medium in the form of magnesium sulfate MgSO ₄ , the concentration of which is 2.2 mmol / L. The characteristics of stable magnesium isotopes existing in nature and their natural content are given in table 1.

Table 1 Isotope Magnetic moment (µv) Natural content,% ²⁴ Mg 0 79 ²⁵ Mg 0.85 10 ²⁶ Mg 0 eleven

For the preparation of sulfates, isotope-friendly oxides ²⁴ MgO, ²⁵ MgO, and ²⁶ MgO produced by FSUE Elektrokhimpribor with a record high isotopic enrichment of 99.8, 98.8, and 97.7 atomic percent, respectively, were used. The passport data of magnesium isotope oxides are given in tables 2-3.

table 2 The content of magnesium isotope, at.% Isotope for ²⁴ MgO for ²⁶ MgO for ²⁵ MgO ²⁴ Mg 99.88 ± 0.02 97.70 ± 0.20 99.37 ± 0.08 ²⁵ Mg 0,07 1.98 0.33 ²⁶ Mg 0.05 0.20 0.30

Table 3 The content of impurities, wt.% Element for ²⁴ MgO for ²⁶ MgO for ²⁵ MgO K <0.005 <Software 0.017 Na 0.002 <Software 0.004 Ca <0.005 0.008 0.34 Fe <0.005 0.019 0,048 Al 0.0011 0,0008 0,031 Si <0.005 <Software <0.005 Cr - <Software 0.0030 Ni 0.0001 <0,0002 <0.0001 Cu 0.0029 0.0021 0,0004 Mn 0.0032 0.0021 0.059 Pb <ON * <Software 0.0015 Lu <Software <Software 0,0003 Pt <Software 0.0031 0,0002 B <Software 0.008 0.0026 Ti <Software <Software 0.0015 Co <Software <Software 0.0011 Sr <Software <Software 0,0002 Ba <Software 0,0003 0,0002 La <Software <Software 0,0003 Eu <Software <Software 0,0002 Zn 0,0006 0,0005 0,0009 Ru <Software 0.0001 <Software Cd <Software 0.0001 <Software P <0.005 <Software <Software

The ratio of magnesium isotopes contained in the nutrient medium M9 was controlled using mass spectrometric analysis. The ratio of magnesium isotopes for M9 media in% are given in table 4.

Table 4 Isotope M9 medium with ²⁴ Mg M9 medium with ²⁵ Mg M9 medium with ²⁶ Mg ²⁴ Mg 99.7 1.4 4,5 ²⁵ Mg 0.12 98.1 0.61 ²⁶ Mg 0.13 0.5 94.9

The thus prepared aqueous solutions were seeded with microorganisms initial concentration of 10 ⁵ cfu / ml (colony forming units per 1 ml). After that, the samples were kept in a thermostat at a temperature of 37 ° C for 10, 13 and 16 hours. The accumulation of cellular biomass was monitored using standard methods for measuring the optical density of a suspension of microorganisms and measuring colony forming units. When bacteria reach a population density of 10 ⁸ -10 ⁹ CFU / ml or 0.6-0.7 rel. units optical density, cell biomass was precipitated by centrifugation at 15 thousand vol. for 15 minutes. After this, the cell biomass of E. coli bacteria was washed twice with distilled water and centrifuged again.

Thus obtained cell biomass was studied for the content of magnesium isotopes by mass spectral (PlasmaQuad 2, VG Elemental, England) and atomic emission methods (ICAP-61, Thermo Jarrell Ash, USA). Cell biomass was previously lyophilized and autoclaved. The data on the ratio of magnesium isotopes in E. coli cells after a culture cycle on M9 isotopic media are shown in Table 5.

Table 5 Isotope Original Culture Tour Exposure in thermostat, hours. Cells grown on M9 medium with ²⁴ Mg Cells grown on M9 medium with ²⁵ Mg Cells grown on M9 medium with ²⁶ Mg 1st water solution 2nd water solution 3rd water solution 1st water solution 2nd water solution 3rd water solution 1st water solution 2nd water solution 3rd water solution ²⁴ Mg 87.8 10 90.8 94.5 95.1 5.9 6.3 6.1 12.5 9.8 9.8 13 91.1 99.5 99,4 6.0 6.6 6.5 10,4 10.0 9.9 16 93.5 99.5 99.5 6.2 6.6 6.5 9.7 10.0 10.0 ²⁵ Mg 5.9 10 6.2 3,5 3.4 90.2 91.9 91.3 2.1 3.3 3,7 13 5.8 0.23 0.22 91.9 92.5 92.1 2.0 1,6 2.6 16 4.7 0.24 0.23 92.3 92.5 92.4 2.2 1,6 1.7 ²⁶ Mg 6.3 10 3.0 2.0 1.4 3.9 1.8 2.6 85,4 86.9 86.5 13 3,1 0.23 1,5 2.1 0.87 1.4 87.6 88.4 87.5 16 1.8 0.22 0.22 1,5 0.87 1,1 88.1 88.4 88.3 * Definition error from 1.2% rel. for 95%; up to 15% for 0.5%

After 10-16 hours of cultivation on magnesium-isotopic media, during which an isotopic exchange between the cells and the aqueous solution took place, the bacteria were enriched in the corresponding magnesium isotope to 99.5 compared to the original bacterial culture grown on Lb nutrient broth, as can be seen from the second column of the data in table 5. The cycle of cultivation of bacteria on nutrient media M9 containing magnesium isotopes leads to almost complete replacement of intracellular magnesium with a specific isotope.

The used method of isotope enrichment of microorganisms in the presence of magnetic and non-magnetic isotopes of magnesium was unique and is used for the first time. This method can be used to obtain isotope-labeled cells of microorganisms, which will find its application in various research works in microbiology, enzymology. In addition, it can be used in various medical and biotechnological applications as a simple and non-laborious way to obtain isotopically enriched biomolecules (ATP, DNA, etc.), cell substructures and the cells themselves. It is important that such isolated molecules and cellular substructures are non-radioactive, since only stable isotopes are used for isotope enrichment. This method can be modified for other stable isotopes of vital chemical elements, as well as for other microorganisms.

Thus, compared with the prototype, the inventive method for isotope enrichment of E. coli cells allows you to effectively carry out up to 90% isotopic exchange between natural magnesium, which is originally contained in the cells, and the magnesium isotope from an aqueous solution.

Claims (1)

A method of enriching E. coli cells with magnesium isotopes, comprising culturing E. coli cells for 10-16 hours at a temperature of 37 ° C in an aqueous solution enriched in magnesium isotope ²⁴ Mg, or ²⁵ Mg, or ²⁶ Mg, including NH ₄ Cl, glucose , Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl and MgSO ₄ , with the following content of components per 1 liter of distilled water:
NH ₄ Cl 2 g MgSO ₄ 200-260 mg glucose (dry) 8-10 g Na ₂ HPO ₄ 11.9-12.1 g KH ₄ PO ₄ 5.95-6.05 g NaCl 0.98-1.02 g