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Transposon Mutagenesis in Rhodococcus sp. 124 Via GentR Transposomes

Supriya Rao
Department of Biology,Massachusetts Institute of Technology, Cambridge, MA 02139
Work conducted in Project Lab 7.13

 
   
Non-Scientific Abstract

Rhodococcus sp. I24 produces several important compounds, some of which may have significance in the pharmaceutical industry.We want to identify the genes that allow the bacteria to make these compounds. One method of identifying genes is transposon mutagenesis. A transposon is a short segment of DNA that randomly inserts into the genome of an organism when introduced, knocking out the gene of function in the process.However, there are no readily available transposons that are functional in Rhodococcus. They lack the proper antibiotic resistance marker needed for selection. In this study, we constructed a transposon that was derived from transposons known to work in other bacterial systems. It carries a gentamicin resistant marker that works in Rhodococcus.We have tested this new transposon and found that it functions in Rhodococcus sp. I24. This new tool will enable the discovery of genes in Rhodococcus.

Abstract

Transposon mutagenesis is a common tool used to study gene function in many microorganisms. The insertion of a transposon into the bacterial genome will disrupt genes and provide insight to their function. However, the absence of an efficient transposon system for use in Rhodococcus has hindered the study of this organism. In order to perform transposon mutagenesis in Rhodococcus sp. I24, a gentamicin resistant (GentR) transposome was constructed. This was done because Rhodococcus sp. I24 has a partial resistance to kanamycin,which is the antiobiotic used in commercially available transposomes. The GentR marker was amplified by PCR inserted into a transposome construction vector to create plasmid pMSR1. In vitro tests of the GentR transposome show that transposition appears to be random. In vivo, transposition into Rhodococcus sp. I24 also appears to be random as judged by preliminary Southern blot data. Since random insertions are needed to study multiple genes, the method of transposon mutagenesis using a GentR transposome will be useful in studying gene function in Rhodococcus sp. I24.