Plasmid maps of pACCNPT10, pACCNPT5.1, pACCl

A. (Top) — Maps of plasmids containing the neomycin phosphotranferase gene (nptH) flanked by regulatory regions from the acetyl-CoA carboxylase gene from C. cryptica. Both plasmids worked well as expression vectors in the diatoms C. cryptica and Navicula saprophila.

B. (Bottom) — Map of plasmid pACCl, containing the full-length genomic sequence of the acetyl-CoA carboxylase gene (accl) from C. cryptica.

Ш — nptII gene sequence; □ — acc1 coding sequence; □ — acc1 regulatory sequences (Source: Dunahay et al. 1995; Roessler and Ohlrogge 1993).

image051

image052image053Replate cells on selective medium

(+ G418)

Pick G418-resistant colonies Test for npfll DNA via Southern Test for NPTII protein via Western

Figure II. B.9. Simplified schematic showing the protocol for transformation of diatoms by microprojectile bombardment (“gene gun”).

image054

Figure II. B.10. Southern blot showing the presence of the nptH gene in transformed cells of

C. cryptica T13L.

Cells of C. cryptica T13L were transformed with pACCNPT5.1 via particle bombardment as described in the text. DNA from wild-type cells (wt) or G418-resistant strains (lanes a-f) were digested with HindIII and hybridized to a digoxigenin-labeled nptll sequence. The lane designated “5.1” contains HindIII-digested pACCNPT5.1as a control. The sizes of DNA fragments included as markers are indicated to the right. (Source: Dunahay et al. 1995).

image055

Figure II. B.11. Western blot, showing the presence of the 30-kDa NPTII protein in transformed cells of C. cryptica T13L and N. saprophila.

In this example, C. cryptica and N. saprophila were transformed with pACCNPT10 and pACCNPT5.1, respectively. Crude cell extracts were separated on SDS-polyacrylamide gels, blotted onto a nitrocellulose filter, and NPTII protein was detected using anti-NPTII primary antibodies and alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibodies. The polyclonal antiNPTII antibody also recognizes a band of approximately 80 kDa in C. cryptica; however, the 30 kDa NPTII protein is seen only in the G418-resistant transformants. (Source: Dunahay et al. 1995).

Добавить комментарий

Ваш e-mail не будет опубликован. Обязательные поля помечены *