Development of three different gene cloning systems for genetic investigation of the new species Amycolatopsis japonicum MG417-CF17, the ethylenediaminedisuccinic acid producer
| Autor: | Stefan Pelzer, Efthimia Stegmann, Wolfgang Wohlleben, Kerstin Wilken |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
DNA
Bacterial Bioengineering Amycolatopsis Molecular cloning Biology medicine.disease_cause Applied Microbiology and Biotechnology Microbiology Polyethylene Glycols chemistry.chemical_compound Plasmid Transformation Genetic Actinomycetales Drug Resistance Bacterial medicine Escherichia coli Bacteriophages Cloning Molecular Cloning Base Sequence Succinates General Medicine Protoplast biology.organism_classification Ethylenediamines Molecular biology Transformation (genetics) chemistry Genes Bacterial Conjugation Genetic DNA Biotechnology Plasmids |
| Zdroj: | Journal of biotechnology. 92(2) |
| ISSN: | 0168-1656 |
| Popis: | For the first time gene cloning systems have been developed for Amycolatopsis japonicum. Direct transformation, polyethyleneglycol (PEG) induced protoplast transformation and conjugal transfer was established for A. japonicum MG417-CF17, the ethylenediaminedisuccinic acid (EDDS) producer. The direct transformation procedure was modified to introduce DNA. The most important parameter for an efficient DNA uptake was the age of the culture. Using of mycelium from 36-h old cultures resulted in the highest transformation frequencies. Further, conditions for transformation of A. japonicum protoplasts were established. The efficiency of transformation depended mainly on the source of PEG and the components of the regeneration agar. The replicative plasmid pULVK2A carrying pA-rep and the apramycin resistance gene was transferred into the EDDS producer with a frequency of 0.38 colonies microg(-1) DNA by using the direct transformation procedure and with a frequency of 0.56 colonies microg(-1) DNA by using the PEG induced protoplast transformation. The plasmid was genetically stable, and could easily be reisolated from A. japonicum. We also demonstrated that conjugal transfer of the plasmid pSET152 from Escherichia coli ET12567 (pUB307) to Amycolatopsis spores is possible. The plasmid pSET152 integrated in the A. japonicum chromosome. A titre of 2.4 x 10(-4) exconjugants per recipient was obtained. |
| Databáze: | OpenAIRE |
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