Part:BBa_K4744000:Experience

The modified LanM gene further was successfully cloned into a plasmid (pYD1).

Cell surface display of LanM without stop codon using pYD1

The LanM gene was amplified with designed primers eliminating the stop codon which enables C-terminal protein fusion and indroducing restriction sites to be able to construct it into the pYD1 vector for cell surface display in S. cerivisiae EBY100. LanM is N-teriminally fused to a subunit of the a-agglutinin receptor AGA-2P and linked to AGA-1P by disulfide bridges. AGA-1P is anchored at the yeast cell surface. The fusion protein also contains a linkerpeptide and an Xpress epitope N-terminally as well as a V5 epitope and a his6-tag C-terminally. The expression is controlled by the promoter GAL1, enabling inducable expression by adding Galactose. Additionally, the vector contains an ori for the replication in E. coli as well as CEN6/ARS4 for replication in S. cerivisiae. The vector contains an ampicillin resistance gene and the gene TRP1 enabling tryptophane synthesis in auxotrophic yeast strains.

It could be proven that cells containing pYD1-LanM were able to adsorb neodymium in significant magnitude compared to cells only containing pYD1, binding up to 64 mg Neodymium per 1 g of cell dry weight.

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