Transformation of E. coli using plasmids Transformation Lab Transformation of E. coli using plasmids

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

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

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

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

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

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

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

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

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

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

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