PCR Detection of the cryD Gene in Recombinant Bacteria and in Microcosm Studies
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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.