Document Type

Thesis - Open Access

Award Date

1984

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Carl A. Westby

Abstract

The science of genetic engineering or direct genetic manipulation required the availability of four basic procedures. These procedures are: i) a way to cut and join DNA molecules, ii) a carrier or vector for genes that can replicate both itself and the foreign DNA it harbors, iii) a way of gaining entrance for this carrier and its foreign DNA into a host cell, and iv) a way of selecting the cells which had received the chimera DNA from among a large cell population which had not. The lac operon of E. coli is a segment of DNA on the chromosome containing three genes designated Z, Y, and A. These are adjacent to a promotor and operator region. The Z gene specifies the amino acid sequence of betagalactosidase. The Y gene specifies the sequence of beta galactosidase permease, and the A gene specifies the amino acid sequence of B-thiogalactosidase acetyltransferase. An I gene, although not part of the lac operon controls it by functioning as a repressor gene. The I gene is transcribed and translated into a repressor protein. This repressor binds to the operator region just downstream from the promotor preventing transcription of the Z, Y, and A genes. The inducer lactose (actually allolactose) if present combines with the repressor at a separate site, changing the configuration of the repressor so that it can no longer bind to the operator. This permits transcription (initiated at the promotor site) and subsequent translation of the Z, Y and A genes. Interest in the E. coli lac operon for cloning in yeast was quite natural owing to the previously widespread use of lac5 (E. coli promotor, operator, and Z gene) as a general indicator of transformation success in E. coli. The lac5 segment produces beta-galactos idaseconstituitively in I E. coli which in the presence of the chromogenic substrate, 5-chloro-4-bromo-3-indolyl-B-D galactoside, (XG), forms vivid blue colonies on agar. Insertions of foreign DNA into lac5 stop production of beta-galactosidase and therefore indicate that insertion of foreign DNA has taken place. Lac operons are currently being used to further modify ColEl derived plasmids carrying the 2um circle DNA. Recently the insertion of an E. coli lac Z gene and Kluyveromyces LAC4 gene into plasmids intended for S. cerevisiae transformation have been described. Purposes of this study were to obtain sufficient lambda lac5 DNA for plasmid insertion. This recombinant plasmid was then to be used for transformation of E. Coli and S. cerevisiae. The ultimate goal was to obtain lactose fermenting strains of S. cerevisiae.

Library of Congress Subject Headings

Genetic transformation
Escherichia coli
Saccharomyce

Format

application/pdf

Number of Pages

126

Publisher

South Dakota State University

Rights

No Copyright - United State
http://rightsstatements.org/vocab/NoC-US/1.0/

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