| Adriamycin (Doxorubicin), a anthracycline antibiotics extracted from Streptomyces peucetius ATCC 29050, is used as antitumor drug in clinical treatment. It can also play an important role in acute lymphoblastic leukemia and myeloid leukemia. Currently,Adriamycin is produced from chemical synthesis and microorganism fermentation which involves a lot of organic solvent,thus synthetic biology streatgy can be an alternative for producing Adriamycin.In this study, the biosynthetic gene cluster of Adriamycin was cloned by PCR amplification of each fragment, restriction enzyme treatment and cloned into cloning vectors.Large fragment was spliced by splicing single fragment via restriction enzyme digestion and ligation. For cloning convenience, the gene cluster was separated into two parts, totally 58 clones. Presently 55 clones were obtained. Once all genes are cloned and spliced into the expression vector,heterlogious expression can be tried to get Adriamycin.Rizobia is a common pat in rhizosphre. The genome sequencing of mode strain Sinorhizobium meliloti Rml021 has been accomplished. To established a fast and concise gene deletion method, we tried the gene deletion through recombineering method.Compared with traditional gene deletion method, Recombineering shows the advanges of simple and highly efficient. We constructed a variety of recombineering helper vectors containing redαβrecombinase expression genes. LacIq was used to replace the lacI for high strigent gene expression. The order of higher strigency is from pLS3304 to pLS3308.The method is firstly prepare the electro-competent S. meliloti Rml021 cells before electroporation of linear targeting DNA generated through OE-PCR. Recombinant is hopefully obtained via kanamycin selection, followed by PCR genotyping analysis and gene sequencing. Unfortunately, no recombinant was obtained after many trials which perhaps due to unsuitable conditions. The present work will lay the foundation for more through recombineering work. |
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