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Fig. 3 | Biotechnology for Biofuels

Fig. 3

From: Genetic engineering of the Calvin cycle toward enhanced photosynthetic CO2 fixation in microalgae

Fig. 3

Generation of transgenic C. vulgaris with enhanced aldolase activity. a Schematic diagram of Synechocystis aldolase expression vector pBI-tpsFBA. sFBA, aldolase from Synechocystis sp. PCC6803; tpsFBA, cTP::sFBA. b Detection of cTP::sFBA gene from the transgenic cell lines of C. vulgaris and wild-type (WT) by PCR analysis. Tps1, Tps2, Tps3, and Tps5, C. vulgaris transformants; M, D2000 marker. c Southern blot analysis of transgenic cell lines of C. vulgaris and WT. Genomic DNA was extracted from transformants and WT and respectively digested with BamHI/SacI and EcoRI, and then electrophoresed on a 0.7% agarose gel. The separated fragments were probed with a DIG-labeled sFBA fragment (333-bp). +, pBI-tpsFBA plasmid as a positive control; M, lambda DNA/HindIII. d RT-PCR analysis of transgenic cell lines of C. vulgaris and WT. Total RNA was isolated from exponentially growing cultures. The RNA quality was determined by the integrity of rRNA bands on agarose gel, and the first cDNA strand was thus prepared. Bands corresponding to the cTP::EGFP cDNA were detectable in all transformants. 18S rRNA was employed as an internal standard. M, 25-bp marker (for PCR) or 10-kb marker (for RNA quality). e Western blot analysis of transformants. Total soluble protein was prepared and separated by SDS-PAGE and then probed with anti-sFBA antibody. M, prestained protein ladder. f Aldolase activity in C. vulgaris transformants and WT. Error bars represent SD (n = 3). Asterisks show significant difference from WT cells (t test, *P < 0.05)

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