In this work, we present the construction of a metagenomic library in Escherichiacoli using the pUC19 vector and environmental DNA directly isolated fromAntarctic topsoil and screened for lipolytic enzymes. Unexpectedly, the screeningon agar supplemented with olive oil and rhodamine B revealed one unusual pinkfluorescent clone (PINKuv) out of 85 000 clones. This clone harbored a plasmid,pPINKuv, which has an insert of 8317 bp that has been completely sequenced.Further analysis of the insert showed eight ORFs. Three ORFs among theseexhibited similarities to Psychrobacter arcticus genes. A nucleotide sequencedesignated as ORF4 encoded a protein with 93% identity to the methylthioadenosinephosphorylase of P. arcticus. This protein was responsible for the observedpink fluorescence of the PINKuv clone in the presence of rhodamine B. We foundthat colonies of recombinant E. coli TOP10F0/pUC19-ORF4 strain showed pinkfluorescence under UV illumination on the Luria-Bertani agar supplementedwith rhodamine B after culturing at 25, 30 and 37 1C. The same effect wasachieved using other E. coli strains such as DH5a, LMG194, JM101 andBL21(DE3) pLysS. The results presented here will provide the basis for furtherstudies on the use of the discovered gene as a new reporter gene for molecularbiology applications.
Authors
- dr hab. inż. Hubert Cieśliński link open in new tab ,
- Anna Długołecka,
- prof. dr hab. Józef Kur link open in new tab ,
- Marianna Turkiewicz
Additional information
- Category
- Publikacja w czasopiśmie
- Type
- artykuł w czasopiśmie wyróżnionym w JCR
- Language
- angielski
- Publication year
- 2009
