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Summer Research Fellowship Programme of India's Science Academies

A MUTATION FROM LEUCINE-60 TO PROLINE ABOLISHES FLUORESCENCE IN GFPmut3

Sheetal. V. Rao

M.Sc Biochemistry, Ramaiah College of Arts, Science and Commerce (RCASC), Bengaluru 560054

Dr. Hemalatha Balaram

Professor, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru 560064

Abstract

Plasmodium berghii is a malarial parasite which infects rodents. Genetic manipulation has had a major impact on the understanding of the Plasmodium genome as it relies on the homologous recombination between a target sequence in the parasite genome and a DNA construct that contains a selection cassette. Recently, methods have been found out to modify the Plasmodium genome efficiently using the lambda Red method of recombineering and also using LR Recombination method. GW-R6K GFPmut3 vector is used for the LR recombination. GFPmut3 has the following mutations-S65G & S72A compared to wild type GFP. Recently, upon sequencing the R6K vector, it was observed that there was a point mutation at 179th position of the GFP nucleotide from T to C. This mutation results in an amino acid change from Leucine to Proline at 60th amino acid. As L60 is conserved across all GFP variants, it is necessary to check if the GFP function is altered. The GFPmut3 is a useful marker for gene expression and as a tag for protein localization. The main objective of this study is to assess the importance of the mutation in the conserved sequence of GFP for its fluorescence properties when cloned and expressed in Rosette strain.

Keywords: Genetic manipulation, Recombination, R6K vector, Leucine, GFP, fluorescence

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