Question

1. (4 pts total) Vibrio cholerae is a naturally competent bacterium. You have 2 strains of V. cholerae: a ciprofloxacin-resistant strain and a ciprofloxacin- sensitive strain. Create a procedure to transform the cipro-sensitive strain with the cipro-resistant DNA over 3 lab periods. Make sure you include reagents, materials, details, and controls. Lab week 1: Lab week 2: Lab week 3:

Answer 1

Lab week 1 : DNA isolation

• isolation of total DNA from resistance strain using DNA isolation kit.
• Fragmentation of DNA into long fragments. (2-3 kb size) using sonication.

Lab week 2 : Transformation

• Inoculate sensitive strain in medium containing fragmented DNA (prepared in lab week 1) and chitin (as V. cholerae gains natural competence upon growth on chitin) in tube #1 (test)
• Inoculate sensitive stain in media containing only chitin (No DNA) in tube #2 (negative control)
• Inoculate resistance strain in media containing only chitin (No DNA) in tube #3 (positive control)
• After incubation of each tube, inoculate from tube #1, #2 and #3 on plate containing ciproflaxacin and do proper labelings.
• Observe growth on each plates after incubation

Lab week 3 : Results

• There should be no growth on plate #2 as No DNA was added nad it is sensitive strain.
• There should be growth on plate #3 as this strain is already resistance to ciproflaxacin.
• If you onserve growth on plate #2, you can say that the transformation of ciproflaxacin resistance gene in sensitive strain was successful.