| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The nucleotide sequences for CDO (NCBI ID: BAE73112) and CSAD (NCBI ID: BAE73113) proteins were obtained for common carp (C. carpio) from the NCBI database. The corresponding nucleotide sequences were codon optimized for expression in C. reinhardtii chloroplast [20,21] as separate genes (cdo::csad), or as fused genes (cdo-csad). The two sequences were then joined with nucleotide sequence coding a GS linker (a 14 amino acid linker; GGGGSSGGGGGGSS) making the fused cdo-csad gene. Codon optimization was performed based on codon usage table for C. reinhardtii chloroplast genome. The codon-optimized cdo-csad (fused with GS linker) nucleotide sequence was synthesized by GenScript USA Inc. The chloroplast transformation vector for C. reinhardtii p322 with aadA cassette (spectinomycin resistance) under 5′ atpA and 3′ rbcL untranslated regions (UTRs) was used for transformation [11]. Sequences homologous to a 5.5 kb region of inverted repeats in the chloroplast genome were targeted for homologous recombination [20,21] (Fig. 2). For the cdo-csad construct, these regions flanked the aadA cassette and the cdo-csad sequence under 5′ and 3′ psbA UTRs, resulting in p322::cdo-csad::aadA construct (Fig. 2b). For the cdo::csad construct, cdo and csadgenes were constructed with spacers to separate the synthetic cdo-csad product by using overlap extension PCR. The regions of homology flanked the aadA cassette, the cdo gene under the influence of 5′ psbA and 3′ atpA UTRs, and csad under 5′ psbDand 3′ psbA UTRs (Fig. 2c). The resultant plasmid was called p322::cdo::csad::aadA. After cloning, the sequences were confirmed by DNA sequencing. | Get A Quote |
Synthetic taurine is an important animal feed supplement critical for fish and cat nutrition. However, there is a demand for natural and renewable sources of this nutraceutical additive. The objective of this study was to increase the production of taurine using genetically engineered Chlamydomonas reinhardtii modified to have the cysteine dioxygenase/cysteine sulfinic acid decarboxylase (CDO/CSAD) taurine synthesis pathway from animals. Because unicellular green microalgae produce taurine at low levels, methods to increase taurine content may qualify the use of these microbes as a feed supplement. Here, increased microalgal taurine content was accomplished using a synthetic approach based on additi... More
