Study On The Heterologous Expression Of Estrogen Degradation Gene In Bacillus Subtilis (DH-BS01) And Its Application

Activated sludge process refers to the artificial ventilation of air into the sewage to produce sludge rich in microorganisms,which can purify the sewage by the action of microorganisms.It has a history of more than one hundred years as a method of biological treatment of sewage.So far,in addition to the common activated sludge process,many improved processes have been derived from the activated sludge process,such as oxidation ditch process,two-stage activated sludge process,sequencing batch activated sludge process and so on.However,there is no report on the improvement of activated sludge process by genetic engineering technology.Therefore,using indigenous bacteria in activated sludge to construct genetic engineering bacteria with specific degradation function to deal with more broad-spectrum pollutants and to enhance the ability of pollutant degradation in activated sludge process is a promising way to improve activated sludge.In this paper,an experimental study is carried out on this assumption.At present,the improvement of strains by genetic engineering has become a relatively mature technology.Genetic engineering refers to the introduction of foreign gene into the recipient cells after recombination in vitro.A biological technique that enables foreign gene to be copied,transcribed,translated and expressed in the recipient cells.The purpose of this paper is to obtain an indigenous strain with independent viability and niche in the indigenous microbial community,prepare it into competent bacteria,and introduce exogenous functional gene by genetic engineering technology to obtain functional strains.The conclusions of this study are as follows:Firstly,a strain was isolated from the activated sludge of the aeration tank of a sewage treatment plant in Changchun.After morphological observation,the colony was round,irregular edge,wrinkled,opaque,expanded,dirty white or slightly yellow,and the bacteria were sticky.Combined with molecular biological identification,the strain was identified as Bacillus subtilis,and named B.subtilis DH-BS01.Secondly,the genomic characteristics of the strain B.subtilis DH-BS01 were obtained by whole genome sequencing analysis.The genome size of the strain was 4.2 Mbp,the GC content was 43.79%,including 4,116 coding genes,213 non-coding RNA,10 gene islands and 4 pre-bacteriophages.The gene function of the strain genome was annotated in multiple databases,a total of 4,090 genes were annotated,and different numbers of genes were annotated in each database.The gene function of the annotation results of each database was analyzed.The comparative genomic analysis was carried out between the strain B.subtilis DH-BS01 and the three reference genomes,and then the exogenous estrogen degradation related gene HSD17B14 was introduced into the vector plasmid pMA5 by means of genetic engineering,and the vector plasmid was transformed into the competent Bacillus subtilis DH-BS01 to construct the recombinant bacteria.Finally,estradiol was selected as the main degradation object of the strain,and the degradation experiments of original Bacillus subtilis,unloaded Bacillus subtilis and recombinant bacteria were carried out with estradiol as the sole carbon source.When the degradation lasted for 72 hours,the degradation rate of original Bacillus subtilis DH-BS01 was 27.2%,and that of unloaded Bacillus subtilis DH-BS01-pMA5 was 21.3%.The degradation rate of estradiol by recombinant strain DH-BS01-pMA5-HSD17B14 reached 57.7%.The experimental results showed that the degradation efficiency of estradiol by genetically engineered bacteria constructed in this experiment was twice as high as that of wild-type Bacillus subtilis.The results of this study enable the genetic engineering strain to obtain a higher efficiency of estrogen degradation than wild-type Bacillus subtilis,which is of great significance to alleviate the estrogen pollution in domestic sewage and aquaculture wastewater and to improve the secondary effluent index of sewage treatment plant.The indigenous strain B.subtilis DH-BS01 of activated sludge has the potential to be a recipient of genetic engineering,which provides a theoretical basis and reliable raw materials for the application of characteristic activated sludge to sewage pollutant treatment.The future research direction is mainly to prepare the indigenous strain B.subtilis DH-BS01 of activated sludge into a bacterial agent with the function of degrading a variety of pollutants through genetic engineering technology,and apply it to activated sludge wastewater treatment in order to improve the efficiency of sewage remediation and improve the ecological environment.