1.3: Expression of the GCL gene was analyzed in bacteria by western blot analysis. The figure shows the Lumi Light mediated light emission of His tagged proteins in a Western Blot analysis. Proteins were purified by Ni-NTA column chromatography, separated on a 10 % SDS-PAGE gel and transferred to a nitrocellulose membrane. His-tagged proteins were detected using an anti-His 5 antibody covalently linked to horseradish peroxidase . Luminescence was recorded on a LAS3000 CCD camera. Lane1 (from right) = His-tagged marker; Lane2 (from right side) = total soluble proteins from induced bacterial culture; Lane3 (from right) = total soluble proteins from non-induced bacterial culture. 

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... full length coding sequence of the GCL gene was amplified by PCR from the genomic DNA of E. coli . The PCR product was cloned into a bacterial expression vector (pET 22b+) (2.1.9) as a N-terminal translational fusion to a His-tag (as described for TSR ). Plasmid DNA isolated from ampicillin-resistant clones was analyzed by both restriction and sequencing. The GCL gene was expressed in the bacterial strain ER2566 in the same way as described for TSR . The soluble proteins from bacterial cells were subjected to Western blot analysis to detect the His-tagged GCL protein. At the beginning, a very faint band was detected meaning that the expression level of the GCL gene in bacteria is very low. The His-tagged GCL protein was purified using Ni-NTA column chromatography and again subjected to western analysis. Figure 3.1.3 shows the result of the western ...

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... Figure 1.1 Schematic representation of the Calvin cycle 3 Figure 1.2 Schematic representation of the NADP-ME type C 4 cycle 4 Figure 1.3 Schematic representation of crassulacean acid metabolism pathway 6 Figure 1.4 Schematic representation of the photorespiratory pathway 9 Figure 2.1 Bacterial expression vector pET 22b(+) 24 Figure 2.2 Bacterial expression vector pTrc99a 27 Figure 2.3 Plant expression vector pTRA-K-rbcS1-cTP 28 Figure 2.4 Plant expression vector withoutchloroplast targeting sequence 30 Figure 3.1 The proposed pathway for the conversion of glycolate to glycerate in 47 the background of the photorespiratory cycle Figure 3.1.1 Expression of TSR gene in bacteria 48 Figure 3.1.2 Measurement of tartronic semialdehyde reductase activity in vitro 50 Figure 3.1.3 Expression of the GCL gene was anylysed in bacteria by western blot 51 analysis Figure 3.1.4 Measurement of glyoxylate carboligase activity in vitro 53 Figure 3.1.5 Expression of the GCL and TSR gene in plants 55 Figure 3.1.6 Expression of both TSR and GCL was detected in transgenic plants 56 Figure 3.1.7 Phenotypic effects of the transgenic plants 57 Figure 3.1.8 Correlation between the expression of the GCL gene and the 58 phenotypic effects observed in the transgenic plant Figure 3.1.9 Phenotypic effects of the transgenic plants expressing TSR or GCL 59 gene Figure 3.1.10 Expression of single TSR or GCL constructs in tobacco 60 Figure 3.1.11 Measurement of glyoxylate carboligase activity in planta 62 Figure 3.2.1 Schematic representation of the construct used for the plastidic 64 transformation Figure 3.2.2 Southern blot results of the chloroplast transformants. 65 Figure 3.3.1 AtGDH shows homology to glycoate dehydrogenases and lactate 67 dehydrogenases Figure 3.3.2 At GDH shows glycolate dehydrogenase avtivity in vitro 69 Figure 3.3.3 Complementation of E. coli glycolate oxidase mutants with At GDH. 71 Figure 3.3.4 Tissue specificity and diurnal rhythm of AtGDH transcript 72 accumulation Figure 3.3.5 Subcellular localization of At GDH. ...