Characterization of psb O mutants from cyanobacterium synechococcus PCC 7942 and expression of the wild-type gene in escherichia coli

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dc.contributor.advisor Vann, Carolyn N. en_US Rosli, Rozita en_US 2011-06-03T19:30:31Z 2011-06-03T19:30:31Z 1994 en_US 1994
dc.identifier LD2489.Z64 1994 .R67 en_US
dc.description.abstract The 33 kilodalton (kD) manganese stabilizing protein (MSP) is intimately associated with the photolysis of water to molecular oxygen. The two main purposes of this study were: 1) to analyze previously constructed MSP mutants and 2) to subclone, express, and purify the wild-type MSP in Escherichia coli in order to investigate the relationship between structure and function of this protein.Growth rates were compared between bacterial cells harboring only the vector, the vector plus the wild-type MSP gene, and the vector plus a mutant MSP gene. No significant differences were seen. This result implies that the expression of the wild-type MSP or mutant MSP is not toxic to the cells. Plasmid DNA isolation and restriction analyses of several of the mutant clones also confirmed the presence of the correct size inserts in the vector. However, upon sequencing several mutant clones, it appeared that losses and/or rearrangements of sequences was occurring. Thus, it was concluded that MSP was not being stably maintained in E. coli.Expression of the wild-type gene was achieved in E. coli by IPTG induction of the gene in pUC120 cloned under the control of the lac promoter. The expressed protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and confirmed by western blotting. Purification of the wild-type protein was obtained by membrane fractionation over a DEAE ion exchange column and the expression product was detected by western blotting. However, the expression product was lost in the concentration procedure and therefore is not available for reconstitution experiments.The wild-type MSP gene was also subcloned in a hybrid shuttle vector pTNTV, previously constructed in our laboratory (1). This construct was used to permit constitutive highlevel expression of the cloned gene and may prove to be an alternative vector to better express the MSP and mutant MSP in future investigations.These results demonstrate that it is possible to express the wild-type MSP gene from cyanobacteria in E. coli, but the problems of instability and recombination of the mutant genes in the vector have to be addressed before proper expression of these genes can be obtained. en_US
dc.description.sponsorship Department of Biology
dc.format.extent viii, 94, [15] leaves : ill. ; 28 cm. en_US
dc.source Virtual Press en_US
dc.subject.lcsh Bacterial genetics. en_US
dc.subject.lcsh Molecular cloning. en_US
dc.subject.lcsh Genetic vectors. en_US
dc.subject.lcsh Cells -- Growth. en_US
dc.subject.lcsh Escherichia coli. en_US
dc.subject.lcsh Cyanobacteria. en_US
dc.title Characterization of psb O mutants from cyanobacterium synechococcus PCC 7942 and expression of the wild-type gene in escherichia coli en_US Thesis (D. Ed.) en_US
dc.identifier.cardcat-url en_US

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  • Doctoral Dissertations [3248]
    Doctoral dissertations submitted to the Graduate School by Ball State University doctoral candidates in partial fulfillment of degree requirements.

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