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|Title:||Studies on the molecular biology of the cyanobacteria Spirulina maxima|
|Authors:||Lee, Clark P.|
|Keywords:||Cyanobacteria -- Biotechnology|
Algae -- Biotechnology
|Abstract:||Early in 1983, we began a study on the cyanobacterium Spirulina sp., stemming from a growing interest in the commercial production of this algae in the Hawaiian islands. The algae is grown primarily as a food supplement due to it's exceptional protein quality. It was proposed that through genetic manipulation, one might be able to further enhance the commercial exploitation of this algae. Although the molecular biology and techniques of genetic engineering in cyanobacteria has advanced rapidly in the past few years, the application to Spirulina has been minimal. The objective of this work was therefore to; 1) generate a shuttle vector and method of mobilization and 2) clone and characterize the phycobiliprotein genes for the purpose of exploiting these high expression, light regulated genes. To start, 15 available strains were screened for endogenous plasmids using the methods in the literature as they developed. The uniform response of the test strains to heavy metal and antibiotic tolerance (specifically to chromium and kanamycine), encouraged the pursuit of all avenues of detection. Unable to detect any plasmids in any strain, an attempt to clone a labile or megaplasmid (or origin of replication) into the E.coli replicating pACYC184 or the Anacystis nidulans shuttle vector pPUC29 (Van denHondel et al.) was investigated. With this "shotgun" library experiments were established to transform the algae and select for the potent chloramphenicol marker. Transformation conditions for cyanobacteria have generated numerous publications and we attempted to repeat most of them using the above mentioned vectors and libraries. The only conditions tested showing any increased survivability was when the algae was grown in iron depleted media and selecting by underagar injection. However, no cultures survived reselection. It became apparent that the presence of a restriction system required investigation. Whitehead et al. has published a procedure for the rapid screening for type II restriction enzymes. Using this method, results show an extremely powerful DNAse is present in Spirulina. Whether this is a restriction system or a nonspecific DNAse is not clear in that we were unable to determine a set of conditions that would produce digestion patterns other than a smear of low molecular weight DNA. This would apparently limit transformation as a method for gene transfer. The alternative method of conjugation has been attempted, however the plating of the mating mixture on mixed media (Spirulina media + 0.5% LB) resulted in mixotrophic lysis of the algae. Finally, in regard to the cloning of ~he phycobiliprotein genes, some unusual problems presented themselves. Using the clone pTP1,and fragments thereof, from Agmenellum quad. (Pilot) as probe DNA, dozens of hybridization conditions were attempted. Only under the conditions of Lemeaux and Grossman (ref.)were we able to obtain definitive signals and only then when the genomic DNA was digested with EcoR1, Bg1II, ClaI and PstI . The explanation for this is not evident from the data obtained in that the base composition, degree of methylation, and C-PC N-terminal amino acid sequence data are not unusual. The stringency conditions required, based on salt and temperature are not unique other than the absence of DNA and protein blocking agents. Further, colony hybridization for library screening was unsuccessful. Only through hybridization of Southern blots of minipreps run on a gel were we able to isolate four unique clones. The clones obtained are from sized Eco/Bgl digests of S.maxima genomic DNA cloned into pBR322 and screened using the alpha structural region of pTP1 as a probe. The restriction patterns and preliminary sequence data for the EcoRI and Bg1II ends are unique to each clone. They are similar in that most sites useful for subcloning into polylinker regions were not detected. Digesting with BstYI(XhoII) gave identical fragments, common to the alpha probe, for every clone however subcloning of these fragments has so far been unsuccessful.|
Thesis (Ph. D.)--University of Hawaii at Manoa, 1989.
Includes bibliographical references (leaves 159-172)
xvii, 172 leaves, bound ill. 29 cm
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|Appears in Collections:||Ph.D. - Biomedical Sciences (Biochemistry)|
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