Method for inhibiting antibiotic resistance gene transformation by using extracellular polymeric substance
An extracellular polymer, antibiotic resistance technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems affecting the transformation process, EPS functional group differences, interaction changes, etc., to reduce the content of ARGs. , the effect of reducing permeability and inhibiting transformation
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Embodiment 1
[0029] Preparation of EPS:
[0030] The selected EPS preparation raw material is the activated sludge of sewage treatment plant.
[0031] First, centrifuge 40 mL of the sludge sample at 8000 rpm for 5 min to remove the supernatant; then clean the sludge, that is, add 10 mL of 0.9% NaCl solution, shake and mix, centrifuge at 8000 rpm for 5 min, repeat the previous cleaning step, remove the supernatant, and retain the sludge ; In addition, add 10 mL of 0.9% NaCl solution, shake evenly, heat at 60 °C for 60 min, and then centrifuge at 10,000 rpm for 10 min; pass the obtained supernatant through a 0.22-μm cellulose acetate membrane, and place the filtrate at -50 °C with a freeze dryer Freeze-dried for 48h to obtain EPS.
[0032] Preparation of competent bacteria and ARGs:
[0033] In this example, Escherichia coli (E.coli trans5α) was used as the competent bacteria, which was purchased from Quanshijin Biological Company, and the LB medium was the E.coli growth substrate during t...
Embodiment 2
[0043] Example 2: Inhibition of conversion by different EPS components
[0044] Preparation of HA, PS and PN:
[0045] HA, PS and PN were simulated with humic acid, sodium alginate and bovine serum albumin, respectively.
[0046] Preparation of competent bacteria and ARGs:
[0047] In this example, Escherichia coli (E.coli trans5α) was used as the competent bacteria, which was purchased from Quanshijin Biological Co., Ltd., and the LB medium was the E.coli growth substrate during the transformation process. ARGs were provided by a plasmid carrying tetracycline resistance (pBR322), purchased from Shanghai Sangon Company.
[0048] LB liquid medium formula: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L;
[0049] LB solid medium formula: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L.
[0050] After the two LB mediums were prepared in the conical flask according to the above formula, the conical flask was placed in an autoclave, sterilized at 121 °C for 20 ...
Embodiment 3
[0060] Example 3: Inhibition of conversion by EPS with different redox states
[0061] Preparation of oxidized and reduced EPS:
[0062] Using a three-electrode cell, EPS was oxidized or reduced at ±0.7V on an electrochemical workstation (CHI600E; CH Instruments, USA) for 12 h, respectively. Platinum sheet and Ag / AgCl were used as counter and reference electrodes, and carbon paper (1 cm × 2 cm) was used as the counter electrode and reference electrode. ) as the working electrode to obtain EPS in oxidized and reduced states, respectively.
[0063] Preparation of competent bacteria and ARGs:
[0064] In this example, Escherichia coli (E.coli trans5α) was used as the competent bacteria, which was purchased from Quanshijin Biological Co., Ltd., and the LB medium was the E.coli growth substrate during the transformation process. The antibiotic resistance gene was provided by a plasmid carrying tetracycline resistance (pBR322), which was purchased from Shanghai Sangong Company.
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