Please use this identifier to cite or link to this item: http://hdl.handle.net/1946/5472
The dim2 ORF from Peltigera membranacea: Amplification and expression cloning
The objective of this project was to clone the dim-2 ORF (open reading frame) from Peltigera membranacea and express it in E. coli in order to produce protein and try to purify it for its study.
The dim-2 gene encodes a methyltransferase enzyme, responsible for DNA methylation.
The dim-2 ORF was amplified by PCR from the genomic DNA of the lichen P.membranacea.
Two oligonucleotides with homology to the ends of the dim-2 ORF were designed including suitable restriction targets (NcoI and NotI) for amplification and subcloning in a pET28a expression vector.
The resulting amplified fragment was purified from an agarose gel and treated with the restriction enzymes NcoI and NotI. The pET28a vector was cut with the same restriction enzymes.
Ligation reactions were performed in a vector insert ratio of 1:3 and transformed into competent E.coli, selecting of kanamacyn plates.
Among the positive transformants, sixteen were selected for PCR analysis using primers contained in the sequence of the insert. This screening was negative and therefore all 700 colonies were screened in 20 pools; again with negative results.
Since no pET28a vector with the dim-2 ORF was recovered, it was not possible to carry out experiments for purification and characterization of the protein.