| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
1. Constraction of a vector : 1)Promoter ; we have searched for promoter-bearing fragments harbored in the chromosomal DNA of Thiobacillus ferrooxidans AP19-3 using a promoter assay vector for Escherichia coli(PMC1403)and have gotten 27 strains having promoter activity. The comparision of the insertion fragments sequences(pTFP12, 15, 19, 26, 28)and the promoter sequence of E. coli(TTGACA -17 bp-TATAAT)revealed high homology among them. As the results of these experiments we concluded that RNA polymerase of E. coli is able to recognize these promoter sequences, resulting in binding to them. 2)Marker gene ; To constract a shuttle vector between E. coli and T. ferrooxidans, a marker plasmid which are able to express in both organisms must be prepared. 3-Isopropylmalate dehydrogenase gene(leuB)which are used in E-coli and Saccharomyces cerevisiae as a marker gene was cloned in E. coli and its sequence was determined. The DNA sequence had high homology with those of E. coli, S. cerevisiae,
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thermophilic bacteria and alkalophilic bacteria. Hereafter, if an ampicillin registance determinant could be inserted into leuB using transposon, this gene can be useful as a marker gene.
2. Preparation of the ampicillin sensitive mutant : We tried to prepare the mutant of T. ferrooxidans AP19-3 sensitive to ampicillin to use it as a host for transformation. 4 mutants sensitive to 25 mug ampicillin/ml were obtained.
3. Examination of the transformation method : Transformations of the prepared plasmid vector into the cells of Thiobacillus by means of the calcium chloride method or the alkaline metal method were very difficult because of their strong cell membrane. We tested the electroporation technique to transfer DNA directly into the cells. Higher levels of membrane permeabilization increased the amount of gene transfer but decreased cell viability. When the cells were treated under 3 puls condtions, cell viabilities were 30-70%. At present, some conditions of the buffer solution for this method have been testing. Less
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