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Fig. 1 | Biotechnology for Biofuels

Fig. 1

From: A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

Fig. 1

Engineered constructs and site of integration of recombinant genes into the C. reinhardtii chloroplast genome. a Schematic diagram of the transformation vectors used to integrate the recombinant genes into the inverted repeat of the chloroplast. The cel6A coding region is regulated by the endogenous atpA promoter, 5′UTR, and terminator in pCHR23, pCHR24, and pCHR25 plasmids; and by the 16S promoter and atpA 5′UTR chimeric construct and the atpA terminator in pCHR72, pCHR73, and pCHR74 plasmids. The cel6A gene is cloned with no added downstream box (DB) in pCHR23 and pCHR72, with the NPTII DB in pCHR24 and pCHR73, and the TetC DB in pCHR25 and pCHR74 plasmids. Primers used to screen for accurate gene insertion and homoplasmicity are indicated by arrows. b DNA agarose gels of PCR products generated using the C. reinhardtii cell lysate and gene-specific primers. The Fwd1 and Rev1 primers were designed around the insertion site. PCR reaction conditions using the Fwd1 and Rev1 primers were chosen to generate amplicons < 500 bp. Of all the tested strains, only the parent wild-type template gave a product, which confirms the homoplasmic state of the engineered strains (top gel). Fwd2 and Rev2 primers were designed within the cel6A sequence and used to confirm transgene integration (second gel from the top). NPTII-Fwd and TetC-Fwd designed within the respective DB sequence and were used in pairs with Rev2 primer to confirm the transgene identity (bottom two gels)

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