1. Transformation of E. coli using plasmids
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 Monocentris japonicus
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?