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Title: A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha

Author
item Solaiman, Daniel
item Swingle, Bryan
item Ashby, Richard - Rick

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2010
Publication Date: 5/10/2010
Citation: Solaiman, D., Swingle, B.M., Ashby, R.D. 2010. A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha. Journal of Microbiological Methods. 82:120-123.

Interpretive Summary: Ralstonia eutropha is a bacterium found in the environment such as soil and sludge. The bacterium is very useful because it can produce biodegradable polymers suitable for replacing petroleum oil-based plastics. The bacterium is also very flexible in terms of what substrates (or “foods”) it can metabolize (or “eat”). As a result, it is also considered for use in removing environmentally harmful chemical pollutants including pesticides. The flexibility of substrate choice allows the bacterium to make biodegradable plastics by using agricultural products such as vegetable oils, animal fats, and corn sugar as starting materials. We therefore are interested in using this organism to convert surplus and inexpensive agricultural products and coproducts into bioplastics. Genetic engineering of the bacterium is necessary to make the process profitable. Currently available methods for genetically modifying Ralstonia eutropha are not ideal because they are either cumbersome or unreliable in ensuring the expression of the new trait. In this report, we described the use of a new DNA molecule (called “vector”) that contains an element (called “promoter”) necessary for ensuring expression of a new trait, to genetically engineer Ralstonia eutropha. Using a green-fluorescent-protein gene as a model, we show that the engineered bacterium can fluoresce (or “glow”). We further showed that an enzyme activity (i.e., alpha-galactosidase) can be expressed in this organism, making it potentially useful for utilizing soy molasses (an agricultural coproduct) to make bioplastics.

Technical Abstract: Ralstonia eutropha is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and the molecular engineering of this organism. We have developed a versatile gene-expression method for the genetic engineering of R. eutropha. This method is based on a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). We successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB'4, with various pBS29-P2-based recombinants using a simplified electroporation protocol. A transformation frequency as high as 4000 Km-resistant colonies/ug DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor alpha-galactosidase in R. eutropha.