BIOL-375 Molecular Genetics

Document Type

Student Paper

Publication Date



Biology | Molecular Genetics

Description, Abstract, or Artist's Statement

The process by which the thermophilic bacteria Meiothermus ruber (M. ruber) synthesizes the amino acid proline is examined in this paper. In the well-studied E. coli system, proline biosynthesis involves three enzymes ProA, ProB and ProC; proA and proB form the proBA operon and both proA and proB possess an upstream Shine-Delgarno sequence; and ProB is functionally dependent on ProA as components of a ProBA complex. In previous studies in Dr. Scott’s lab, the putative M. ruber proA (locus tag Mrub1079), proB (Mrub1080) and proC (Mrub1345) genes were cloned into the pKt1 expression vector, transformed into the corresponding E. coli null strains, and tested for complementation. In addition, an Mrub1080-1079 clone was tested for complementation against E. coli proA - and proB - null strains. The M. ruber gene Mrub1345 successfully complemented E. coli proC - null strain but complementation of Mrub1079, Mrub1080 and Mrub1080-1079 clones produced inconclusive results. In this paper, we demonstrate conclusively that Mrub1079 and Mrub1080 complement their respective E. coli null strains, thereby confirming the function of the M. ruber genes in proline biosynthesis. The outcome of the complementation test was not enhanced by testing an intact proBA operon, or by adding an E. coli version of a Shine-Delgarno sequence upstream of the Mrub1079 in the Mrub1080-1079 clone.