Chromosomal and Extrachromosomal Integration of the Green Fluorescent Protein (GFP) Coding Region in Rhizobium

News source: Center Research for Biotechnology,
Indonesian Institute of Science
For further information, contact Nurwantara, S.K., Sukiman H.I., and Ariani, D. Center Research for Biotechnology, Jalan Raya Bogor Km.46, Cibinong 16911, Indonesia.
Phone: 21-8754587, Fax: 21-8754588
Email: witarto@biotek-indonesia.net, 2003-02-01

Two Variants

TWO VARIANTS of the green fluorescent protein (GFP) coding regions were introduced into Rhizobium and Bradyrhizobium cells. The first variant, gfpuv with a hybrid promoter harboured in pBBR-GFPUV, was introduced into Rhizobium by means of electrophoration. The second variant, egfp containing Ser65-Thr and Phe64-Leu double mutation, was transferred into the rhizobial genome by transposomal conjugation of pUT-Tn5 egfp-accCi-KmF (Ap^R) in E. coli S-17 g-pir as donor.

Best Result

The best result was obtained from Rhizobium DBM-53 under the following electrophoration condition: voltage, 2.5 kV; capacitance, 25 mF; resistance, 360-720w; and pulse for 30 msec. Integration of Tn5 egfp-accCl-KmF resulted in Nx^R, Km^R, and Gm^R transconjugants which expressed the GFP from their own promoters. The integration stability of the two variants of GFP in Rhizobium DBM-53 will be studied under laboratory and greenhouse conditions.

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