Plasmid Transformation in E. coli: Gene Incorporation and Antibiotic Resistance

1. Transformations

2. Background • Transformation is the incorporation of new genes into a cell from DNA that the cell takes up from its environment • Occurs naturally, but captured and widely-used for technology, etc. • Plasmid is a small, circular DNA molecule that replicates independently of the much larger bacterial chromosome

3. pVIB • Contains the lux genes: enable the host cell to bioluminesce; derived from Vibrio fisheri, a bioluminescent bacterium • Contains a gene conferring resistance to the antibiotic ampicillin

4. Ampicillin-sensitive cells in medium + ampicillin • no growth • Ampicillin-resistant cells in medium + ampicillin • growth • Use of a selectable marker to obtain E.coli cells transformed by pVIB • E. coli cells (untransformed): ampicillin-sensitive • E. coli cells carrying pVIB: ampicillin-resistant • Selecting for ampicillin resistance selects for cells that have been transformed.

5. Approach • +plasmid suspension on LB + amp: • Experimental • –plasmid suspension on LB + amp: • negative control • + plasmid suspension on LB: • positive control • –plasmid suspension on LB: • positive control

6. Really Quick Overview of Plasmid Lab • Plasmid tube, non-plasmid tube (with CaCl2) • + Ec in both, mix until white • + plasmid DNA into “Plasmid” tube • Incubate both for 15 min • Label plates • Heat shock cells • into 42C for 90 sec, agitate • Back to ice for 1+ min • +250 ml LB to both tubes, gently mix, rest 15+ min • Spread cells on appropriate plates • Rest 15, flip plates, incubate 24+ hours