A kind of utilize three parent's pairings to engage and with petroleum hydrocarbon be sole carbon source screening carry petroleum hydrocarbon degradation gene can from the method for transfer broad host range plasmid

A kind of utilize three parent's pairings to engage and with petroleum hydrocarbon be sole carbon source screening carry petroleum hydrocarbon degradation gene can from the method for transfer broad host range plasmid

  • CN 102,517,279 B
  • Filed: 12/23/2011
  • Issued: 03/23/2016
  • Est. Priority Date: 12/23/2011
  • Status: Active Grant
First Claim
Patent Images

1. one kind utilize three parent'"'"'s pairings to engage and with petroleum hydrocarbon be sole carbon source screening carry petroleum hydrocarbon degradation gene can from the method for transfer broad host range plasmid, it is characterized in that:

  • select the karyomit(e) of different Proteobacteria subclasses with selective marker recipient bacterium with carry and oneself can not shift (Tra-Mob+) and donor bacterium with selective marker plasmid, engage by matching with sample three parent, the plasmid of donor bacterium in the sample to which can enter recipient bacterium under the assistance of rotation (Tra+Mob+) broad host range plasmid, the selection markers that zygote is had by recipient bacterium and donor cingula is screened, again by screening containing cultivating in the substratum taking petroleum hydrocarbon as sole carbon source the zygote of rotation broad host range plasmid, screening can the broad host range plasmid of decomposing petroleum hydrocarbon,Described screening method is specially;

    1) microbial cell suspension is prepared;

    get testing sample and add in physiological saline and shake on shaking table, then left standstill by suspension, get supernatant liquor and carry out centrifugal;

    It is resuspended that precipitation is placed in LB liquid nutrient medium, and abundant eddy current is stand-by;

    2) activation of donor bacterium and recipient bacterium and liquid culture;

    respectively by frozen carry can not oneself shift (Tra-Mob+) and donor bacterium with selective marker plasmid and karyomit(e) with recipient bacterium incubated overnight at the LB solid medium containing corresponding selective marker 30 DEG C of selective marker;

    Then above-mentioned donor bacterium, the single bacterium colony of recipient bacterium are inoculated in not containing in the LB liquid nutrient medium of selective marker respectively, and 30 DEG C of incubator overnight are cultivated, stand-by;

    3) three parent'"'"'s pairings;

    with the LB nutrient solution of dilution 10 times by step

         2) in grow sufficient donor bacterium and recipient bacterium nutrient solution is diluted to 10 respectively -2doubly;

    After getting above-mentioned dilution respectively, donor bacterium and recipient bacterium mix with microbial cell suspension, then centrifugal and by the resuspended above-mentioned precipitation of 1/10LB liquid nutrient medium, and resuspended rear nutrient solution to drop in LB solid medium incubated overnight at 30 DEG C, stand-by;

    4) zygote screening;

    scraping step

         3) the middle lawn grown, in stroke-physiological saline solution, resuspended culture, is aseptically diluted to 10 by stroke-physiological saline solution -4, getting 0.1ml extent of dilution is 10 0-10 -1the LB solid medium of above-mentioned resuspended culture coating containing two selective markers of the selective marker of recipient bacterium and the selective marker of donor bacteria plasmid in, be placed in 30 DEG C of constant incubators, incubated overnight;

    5) plasmid preparation and diversity checking in a small amount;

    picking step

         4) middle zygote list bacterium colony, access in the above-mentioned LB liquid nutrient medium containing two selective marker, 30 DEG C of incubator overnight are cultivated;

    SDS-alkaline hydrolysis is then adopted to extract zygosporic plasmid, and by agarose gel electrophoresis, screening broad host range plasmid;

    6) plasmid petroleum hydrocarbon metabolic function checking;

    above-mentioned gained zygote is inoculated in petroleum hydrocarbon be sole carbon source liquid inorganic salt culture medium in cultivate, have in the zygote liquid medium within of petroleum hydrocarbon degradation ability and can show growth, treat that in nutrient solution, substratum becomes muddy, OD600 considerable change, being that this zygote contains with petroleum hydrocarbon is wide host'"'"'s petroleum hydrocarbon metabolism plasmid of sole carbon source;

    Or by above-mentioned gained zygote dibbling 30 DEG C of cultivations on the solid double-layer flat board being sole carbon source with polycyclic aromatic hydrocarbons component, have in the zygote liquid medium within of petroleum hydrocarbon degradation ability and can show growth, and by solid plate under ultraviolet light, the absorption under ultraviolet light of periphery of bacterial colonies substratum weakens, there is the dark circle of degraded in periphery of bacterial colonies, namely this zygote contains with polycyclic aromatic hydrocarbons is wide host'"'"'s petroleum hydrocarbon metabolism plasmid of sole carbon source;

    Described donor bacterium is the plasmid-encoded chlorampenicol resistant of E.coliJM109pBBR1MCS, the plasmid-encoded gentamicin resistance of E.coliJM109pBBR1MCS-5 or the plasmid-encoded chlorampenicol resistant of E.coliDH5pSU4814;

    Recipient bacterium is E.coliK12rif rifampicin resistance, E.coliK12nal nalidixic acid-resistant, P.putidaUW3 rifampicin resistance or C.necatorJMP228 rifampicin resistance.

View all claims
    ×
    ×

    Thank you for your feedback

    ×
    ×