Molecular Biology Experiments Utilizing the lux Genes of Vibrio fischeri and gfp Gene of 
     Aequoria victoria

• Introduction
  
  
• Lux Bioluminescence
  
  
• GFP System
  
  
• Bacterial Transformation Experiment
  
  
• PCR Amplification of luxR Gene and Ligation into Plasmid Vectors
  
  
• Comparison of Lux and GFP Systems
  
  
• SDM of Dark Mutation of luxRGene
  
  
• RT -PCR
  
  
• Southern and Northern Blotting Made Easy
  
  
• Obtaining Cultures and Plasmids for Experiments
  
  
• References
  
  
• Links
  
  
• Contact Information

 
                                                       The Lux Logo:
                        

Introduction

This webpage describes a series of laboratory exercises utilizing the luxR gene of Vibrio fischeri and the gfp gene of Aequoria victoria  to teach the broad applications of PCR (polymerase chain reaction). PCR is a powerful tool in the arsenal of recombinant DNA techniques. PCR is an in vitro reaction that allows a specific sequence of DNA to be amplified into millions of copies within a few hours.The technique involves the selection of two different primers (oligonucleotides) that are complementary to the two ends of the target DNA sequence.

Laboratory experiments are needed to teach this technology to students. Many PCR experiments only amplify a segment of DNA and cloning experiments only involve the cloning and expression of an antibiotic resistance gene. In these cloning experiments, students begin with E. coli colonies on an agar plate and end with identical looking colonies. While these experiments are successful and instructive, they lack the wonderment that is the essence of genetic engineering. When PCR is combined with the lux bioluminescence and/or the green fluorescence protein (gfp) systems, students perform powerful and exciting experiments. The specific experiments described in this webpage include:
                          -Bacterial transformation
                          -Directional cloning of the luxR gene
                          -Site directed mutagenesis (SDM).
                   -RT-PCR of the luxR gene of Vibrio fischeri
                          -Southern and Northern Blotting Made Easy

The bacterial transformation experiment is a basic experiment that can be performed in an introductory biology course. The PCR experiments are advanced techniques suited for a college level molecular level biology course. ^top

©2008  James Slock, Ph.D
Biology Department,  King's College
133 N. River Street, Wilkes Barre, PA 18711
Phone: (570) 208-5900 ext. 5724  Fax: (570) 208-6024
Email: jaslock@kings.edu
Designed by: King's College Mass Communications/ Media Technologies Department
Designed: Spring 2000