Transformation Lab

1. Transformation Lab Transformation of E. coli using plasmids

2. What we will see today (and in two days…) • Bacteria contain circular DNA pieces called plasmids • Bacteria can take in plasmids from other bacteria • Bacteria can take in plasmids that contain genes from other sources

3. Background and Review • A gene is a piece of DNA that contains the instructions for making a particular protein; this protein gives an organism a certain trait • Some genes can be inserted into organisms to change their traits; this is called . . . TRANSFORMATION

4. Anatomy of a Bacteria Cell • Bacteria contain bits of circular DNA called plasmids • Bacteria can exchange plasmids freely with one another and with other sources

5. What plasmid are we using and why? • pVIB plasmid will be used • This plasmid contains • A gene for ampicillin resistance • A gene to allow the colonies to glow in the dark

6. Where does this glow in the dark gene come from? • We will use the “glow gene” of Vibrio fisheri(glow in the dark bacteria) that lives in the light organ of Monocentrisjaponicus • This is composed of many genes – all together called the lux gene • Gene for production of luciferase enzyme • Gene for production of aldehyde – action of luciferase on the aldehyde produces the glow • Regulator gene

8. You will use… • Two different experimental set ups: • One plate of nutrient agar • Two plates of agar containing ampicillin • You will create cultures: • One containing the plasmid • One without the plasmid

9. Experimental Set Up • Half of the groups will plate “-plasmid” culture on the LB nutrient agar (without ampicillin) • Half of the groups will plate “+plasmid” culture on LB nutrient agar (without ampicillin) • ALL GROUPS WILL PLATE: • +plasmid on LB/Amp plate • -plasmid on LB/Amp plate

10. Important points • Label EVERYTHING • Name • Date • Contents (LB vs. LB/Amp; + vs. – plasmid)

11. Review Questions On which of the plates would you expect to find bacteria most like the original non-transformed E. coli on your starter plates? Explain your predictions. LB –plasmid LB/amp –plasmid LB/amp +plasmid

12. Review Questions If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions. LB –plasmid LB/amp –plasmid LB/amp +plasmid

13. Review Questions What is meant by a control plate? What purpose does a control serve?