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Poster Presentation 2-20
Gene Transfer and Green Fluorescent Protein (GFP) Expression in Gram-negative Ethanol Tolerant Thermophiles
Kenlee Nakasugi, Charles J. Svenson, Nicholas J. C. Fong, Dianne R. Glenn and Peter L. Rogers
Department of Biotechnology, University of New South Wales Samuls Building, Botany Street, Randwick, Sydney, New South Wales 2052, Australia
Telephone: 61-2-9385-3896; Fax: 61-2-9313-6710; E-mail: p.rogers@unsw.edu.au
Ethanol production by thermophilic bacteria has a number of potential advantages over current mesophilic ethanol production processes. These include a reduction in energy costs during fermentation, broad substrate range, and easy recovery of volatile products. However, in order to realize these benefits, pyruvate must be redirected from lactate production to ethanol. To achieve these goals, target thermophilic bacteria need to be amenable to genetic manipulation.
Results from DNA transfer experiments in four newly isolated, Gram-negative, ethanol tolerant thermophilic facultative anaerobic bacteria are reported. Exconjugants were consistently obtained for all strains in tri-parental conjugations experiments with Escherichia coli strains DH5a:pCJS10 (donor) and C600:pRK600 (helper). Chloramphenicol acetyltransferase (cat) and Green Fluorescent Protein (gfp) were used as selectable markers. Two of the exconjugant strains fluoresced when observed by epi-fluorescence microscopy (excitation 495nm emission 520nm). Electroporation investigations yielded electrotransformants for two of the strains. To our knowledge this is the first reported instance of conjugation between a mesophile and thermophile and the first report of GFP expression in vivo at 60°C.
This RFS1010 based vector is currently being used to assess 42 native promoters isolated from these new bacteria. Further, work is progressing on the redirection of pyruvate from lactate production to ethanol by the introduction of genes encoding for thermostable pyruvate decarboxylase and the deletion of ldh.
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