Chitin-Mediated Bacterial Transformation

Natural transformation experiments on crab shell fragments were performed as described [8 (link),9 (link)]. Variations thereof were used in order to test different chitin/chitin derivative sources: V. cholerae A1552 cells were grown at 30°C until an OD₆₀₀ of approximately 0.5, washed and resuspended in DASW or M9 medium. Autoclaved chitin flakes, chitin powder or chitosan (50-80 mg each) were subsequently inoculated with 0.5 ml washed bacterial culture plus 0.5 ml fresh medium, mixed thoroughly and incubated at 30°C for 16-20 hours. After exchange of the medium (except where indicated) donor DNA was added as transforming material. The DNA was either gDNA of strain A1552-LacZ-Kan (positive control) or PCR-derived DNA as explained in the text. Cells were further incubated for either 2 hours (expedite protocol) or 24 hours (standard protocol), respectively, and subsequently detached from the chitin surface by vigorously vortexing for 30 sec. Transformants were selected on LB + Kanamycin (75 μg ml^-1) plates and transformation frequencies were scored as number of Kanamycin-resistant CFUs/total number of CFUs. Chitin and chitin derivatives used in this study: Chitosan (Fluka; cat. # 448869), Chitin flakes (Sigma; cat. #C9213), Chitin powder (Sigma; cat. # C7170) and Dungeness crab shells (Fisherman's Wharf, San Francisco, CA).