PCR Detection of the cryD Gene in Recombinant Bacteria and in Microcosm Studies
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Authors
Giroux, Janna
Issue Date
1997
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
The cryD gene sequence, natively found in plasmids of Bacillus thuringiensis
israelensis (Bti), codes for a parasporal crystal protein (CryO) that is toxic to larval
mosquitoes and other insects of the Order Diptera. Studies of this bacterium are essential to develop biological control of mosquitoes and black flies as vectors of diseases. A polymerase chain reaction (PCR) protocol was developed to amplify this gene sequence which was then visualized on an agarose gel. A band size of 788 bp confirms the presence of cryD. Using this protocol, the presence of CryD was detected in plasmids found in Bti and other recombinant bacterial species, including Bacillus megaterium and Escherichia coli. Plasmids were extracted using either the Wizard Kit or the alkalinelysis technique.
Microcosms are used as models that portray some features of the natural
environment. In this study, a standard concentration of bacterial cells was inoculated into
larval mosquito habitat water. At specific times, the bacteria were isolated from the
microcosms and their plasmids extracted. The plasmids underwent PCR amplification
and cryD was visualized on a gel, indicating stability, or not, indicating absence.
Escherichia coli was the only bacterial species used in which cryD was visualized
on the gel. The appearance of the gene confirmed that the bacteria can not only survive,
but also reproduce in the nutrient poor water. In contrast, counts from spread plates
made at the time of plasmid extractions showed that Bti decreased in colony numbers in
the microcosm water after 3 1/2 hours.
Description
v, 22 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.