New method for breeding strains of modern microbial genome technology

New method for breeding strains of modern microbial genome technology

1    Gene Chip ferrous oxide acidophilic Thiobacillus pick test method and activities

Acidophilic Thiobacillus ferrooxidans (Acidithiobacillus ferrooxidans, hereinafter referred to as A .f bacteria), is the most commonly used biological metallurgical bacteria, but also the most studied bioleaching microorganisms. Using the A.f standard strain ATCC 23270 genome-wide 3217 gene sequence information, the A.f standard strain genome-wide gene chip was constructed. Through the sowing bottle test, column leaching test and heap leaching test of sulfide ore in different mining areas in China, A.f strains with different oxidation activity types were screened. At the same time, the genomic DNA and RNA were extracted and purified from the sample strain and the model strain and fluorescently labeled; the fluorescent label product of the sample with the same amount of labeling and the fluorescent label product of the model strain were mixed, and the whole genome of the constructed A.f standard strain was constructed. The gene chip was hybridized for 12-18 h; the hybridized chip was scanned with a laser copolymerization scanner at a resolution of 10 μm. Through the above experimental analysis, 967 genes were found to be unique to different strains in the 3,217 genes, and the remaining 2,250 genes were shared by all A.f bacteria. Another 320 high-oxidation-active bacteria characteristic genes were found. National standard for the detection of Thiobacillus ferrooxidans and its active gene chip (GB/T 20929-2007), and from Guangxi Dachang, Yunnan Dongchuan, Simao, Guangdong Dabaoshan, Meizhou, Hubei Daye, 20 is typically a plurality of mine Dexing, Gansu white silver, Liuyang Qibaoshan acidic mine water leaching or heap effusion cell sampling parts 120, discussed above analysis recite height 5 activity detected from the sample a .f The strain was identified as the acidophilic Thiobacillus ferrooxidans after analysis of culture traits, morphology, physiological and biochemical characteristics and 16S rDNA sequence. Further on-site leaching test found that after 75 days of leaching, the leaching rate reached 65.25%, the standard strain ATCC 23270 was 37.54%, and the other strain of low-activity bacteria was only 24.86%.

2 flora bioleaching rapid detection of microbial community genome arrays

The pure genomic DNA obtained by the isolated culture was directly spotted on the chip substrate to prepare a immersion microbial community genomic chip. The community genomic chip contains 5 genera, 15 species, and more than 50 strains. The results showed that under the hybridization conditions of 55 °C and 50% formamide, the chip has interspecific specificity, and its sensitivity reaches 0.11ng genomic DNA, and has good quantitative performance. The chip can simultaneously detect the leaching environment. A variety of microorganisms use this information to provide genetic guidance for the selection of leaching strains.
3 flora quick analysis of the structure and function of bioleaching microbial functional gene chips

The so-called functional gene chip is a gene chip constructed by using a functional gene related to physiological activities as a probe. At present, the functional gene chip for the analysis of the structure and function of the leaching microbial community, in addition to the functional gene, also contains the 165 rRNA gene of the leaching microorganism which can reflect the development and taxonomic status of the microorganism. Among them, 825 functional gene sequences related to the leaching process were obtained from 38 microorganisms and 518 clones, and 603 probes were designed to obtain 16S rRNA genes from microorganisms and clones related to the leaching system. Sequence 2003, and designed 600 probes. The chip is capable of combining leaching microbes 16S rRNA and functional genes to simultaneously analyze the presence of microorganisms in the environment and the possible role of these microorganisms, thereby using this information to purposefully screen for specific functions of leaching microorganisms.

4 bioleaching microbial flora metabolism genomic analysis chip Acer

X.Guo, who use copper ore leaching environment DEXING total DNA extracted mining system constructed to give a metagenomic library, the metagenomic library comprising 7,776 clones CCP 30 ~ 50kb containing large insert. At the same time, in order to rapidly screen the clone containing the gene of interest from the library, a macrogen chip was constructed using the library. On the other hand, the researchers also screened a novel nitrogen-fixing gene cluster from a metagenomic library by chip hybridization. Further analysis revealed that the gene cluster is completely different from the gene cluster in any of the known nitrogen-fixing microorganisms, which is likely to come from an unknown extremely acidic microorganism. In the course of these studies, a deeper understanding of the microbial community genetics and metabolic processes in the leaching system has provided a good basis for further research. This study shows that metagenomic microarray as a high-throughput genomic tool to study the metabolic diversity of microbial communities has potential application value, which is of great significance for screening efficient strains.

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