Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Chloroplast expression vector and PCR analysis. (A) pLD-BD Cry2Aa2 operon (9.8 kb) with PCR primer binding sites and expected fragment sizes. PCR analysis of untransformed and putative chloroplast transformants using two primer sets: (B) 1P1M and (C) 3P3M. Lane 1, 1 kb ladder; lane 2, untransformed; lanes 3–7, pLD-BD Cry2Aa2 operon putative transformants; lane 8, pLD-BD Cry2Aa2 operon plasmid DNA.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Expressing, Plasmid Preparation, Binding Assay

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: 10% SDS–PAGE gel stained with R-250 Coomassie blue. Loaded protein concentrations are provided in parentheses. Lane 1, prestained protein standard; lane 2, partially purified Cry2Aa2 protein from E. coli (5 μg); lane 3, single gene-derived Cry2Aa2 pellet extract solubilized in 50 mM NaOH (22.4 μg); lane 4, single gene-derived Cry2Aa2 supernatant (66.5 μg); lane 5, operon-derived Cry2Aa2 pellet extract solubilized in 50 mM NaOH (22.9 μg); lane 6, operon-derived Cry2Aa2 supernatant (58.6 μg); lane 7, untransformed tobacco pellet extract solubilized in 50 mM NaOH (29.8 μg); lane 8, untransformed tobacco supernatant (30.4 μg). Colored compounds observed in the supernatant of transgenic plants interfered with the DC Bio-Rad protein assays.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: SDS Page, Staining, Purification, Derivative Assay, Transgenic Assay

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Transmission electron micrographs. Operon-derived Cry2Aa2 leaf sections in young (A), mature (B, D), and old, bleached leaf (C). (E) Single gene-derived Cry2Aa2 mature leaf; (F) mature untransformed leaf.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Transmission Assay, Derivative Assay

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Protein quantification by ELISA in young, mature, and old transgenic leaves. (A) Single gene-derived Cry2Aa2 expression shown as a percentage of total soluble protein. (B) Operon-derived Cry2Aa2 expression shown as a percentage of total soluble protein.

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Enzyme-linked Immunosorbent Assay, Transgenic Assay, Derivative Assay, Expressing

Journal: Nature biotechnology

Article Title: Overexpression of the Bt cry 2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

doi: 10.1038/83559

Figure Lengend Snippet: Insect bioassays. (A, D, G) Untransformed tobacco leaves; (B, E, H) single gene-derived Cry2Aa2 transformed leaves; (C, F, I) operon-derived Cry2Aa2 transformed leaves. (A–C) Bioassays with Heliothis virescens; (D–F) bioassays with Helicoverpa zea; (G–I) bioassays with Spodoptera exigua. All leaf samples for each replicate were from the same leaf. Two samples were evaluated per treatment, and observed daily for mortality and leaf damage for five days. Treatments were replicated three times. Insects were tested at 5 or 10 days old (see text for details).

Article Snippet: Protein extraction buffer from the Cry2Aa2 plate kit from Envirologix (Portland, ME) used for quantification was added to the powder, and further grinding was done.

Techniques: Derivative Assay, Transformation Assay