Guy Caldwell & Shelli Williams 
Integrated Genomics 
A Discovery-Based Laboratory Course

Integrated Genomics: A Discovery-Based Laboratory
Course introduces the excitement of discovery to the
basic molecular biology laboratory. Utilizing up-to-date molecular
biology protocols and a basic experimental design, this text offers
experience with three different model systems. Students will become
familiar with the simplicity and power of single-celled organisms,
Escherichia coli and Saccharomyces cerevisiae, as
they search for genes that interact and function within the
nematode Caenorhabditis elegans.

Incorporated throughout the course are exercises designed to
offer students familiarity with the wealth of bioinformatics data
that can be accessed on the World Wide Web. Following completion of
interaction studies within the yeast, the course is designed to
allow students to examine the functional consequences of reducing a
gene’s function within the multicellular worm that is both
simple and inexpensive to maintain within a laboratory. The
inclusion of alternative experiments allow for flexibility in
determining the ending date or goal of the laboratory, as well as
working within the available budget and resources of most any
classroom environment.

Further striking features of this title are:

* An accompanying Web site providing Power Point slides, plus
links to the internet, and regular updates as bioinformatics
databases evolve and methods improve. href=’http://www.wiley.com/go/caldwell’>www.wiley.com/go/caldwell

* Inclusion of modern genomic/proteomic technologies such as the
yeast two-hybrid system and RNAi

* Detailed experimental protocols and easy access to
instructional materials

This discovery-based laboratory course provides excellent
practical training for those pursuing career paths in biomedicine,
pharmacy, and biotechnology.

Table of Content

Preface.

Author biographies.

Acknowledgments.

List of figures.

1 Introduction to basic laboratory genetics.

1.1 Transferring and handling C. elegans.

1.2 Introduction to laboratory genetics.

2 Gene expression analysis using transgenic animals.

2.1 Transgenic gene expression analysis in C. elegans: lac Z staining.

2.2 Transgenic gene expression analysis in C. elegans: GFP analysis.

3 Creation and testing of transgenic yeast for use in protein-protein interaction screening.

3.1 Small-scale transformation of S. cerevisiae.

3.2 Transformation of S. cerevisiae to test for non-specific interaction.

3.3 Assaying for protein-protein interaction by reporter gene expression.

4 Yeast two-hybrid screening.

4.1 Protein-protein interaction screening of a C. elegans c DNA library.

4.2 Assaying for protein-protein interaction by reporter gene expression.

5 Isolation and identification of interacting proteins.

5.1 Preparation of electrocompetent E. coli.

5.2 Isolation of DNA from yeast and electroporation of E. coli.

5.3 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.

5.4 Sequencing of two-hybrid library plasmid DNA vectors.

6 Using bioinformatics in modern science.

6.1 DNA sequence chromatogram.

6.2 BLASTing your sequence.

6.3 Evaluating sequence results and choosing an RNAi target.

6.4 Bioinformatics practice questions.

7 Generation of an RNAi vector.

7.1 Small-scale isolation of genomic DNA from C. elegans.

7.2 PCR amplification of target gene sequence from C. elegans genomic DNA.

7.3 Preparations for cloning to generate RNAi vector.

7.3.1 Agarose gel electrophoresis.

7.3.2 Removal of d NTPs from PCR reaction.

7.3.3 Restriction enzyme digestion of PCR product and C. elegans RNAi vector.

7.4 Gel purification of DNA and ligation of vector and PCR-amplified DNA.

7.4.1 Preparative agarose gel electrophoresis.

7.4.2 Gel purification of DNA from agarose gel.

7.4.3 Ligation of vector and PCR-amplified DNA.

7.5 Transformation of ligation reactions.

7.6 PCR screening of transformation colonies.

7.7 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.

7.8 Verifying successful ligation by restriction digestion.

8 RNA-mediated interference by bacterial feeding.

8.1 Preparation of RNAi-feeding bacteria for transformation.

8.2 Media preparation for RNAi feeding.

8.3 Transformation of RNAi-feeding strain HT115(DE3).

8.4 RNA interference by bacterial feeding of C. elegans.

8.5 Analyzing effects of ds RNAi.

8.5.1 Assaying for sterility (Ste) or embryonic lethality (Emb).

8.5.2 Assaying for growth effect.

8.5.3 Assaying for morphological effects.

8.5.4 Assaying for general neuromuscular effects.

8.5.5 Assaying for specific neuronal effects.

8.5.6 Assaying for dauer formation.

Appendix I Recombinational cloning.

AI.1 Isolation of genomic DNA from C. elegans.

AI.2 PCR amplification of target gene sequence from C. elegans genomic DNA.

AI.3 Agarose gel electrophoresis and clean-up of PCR reaction.

AI.4 Entry vector cloning.

AI.5 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.

AI.6 Destination vector cloning.

AI.7 Small-scale isolation of plasmid DNA from E. coli: the mini-prep.

Appendix II Recipes and media preparation.

Solution recipes.

Media preparation.

Appendix III Sterile techniques and worm protocols.

Sterile techniques.

Worm protocols.

Appendix IV Mutant C. elegans phenotypes.

Appendix V Vector maps.

Subject index.

About the author

Guy A. Caldwell, Ph.D., is an Assistant Professor in the
Department of Biological Sciences at The University of Alabama in
Tuscaloosa, where since 1999, he has held an undergraduate
professorial appointment from the Howard Hughes Medical
Institute. In 2001, Dr. Caldwell was named a Basil O’Connor
Scholar of The March of Dimes Birth Defects Foundation for his
research into the molecular basis of childhood birth defects of the
brain. Dr. Caldwell is a recipient of grants from The March of
Dimes, National Institutes of Health, the Dystonia Medical Research
Foundation, American Parkinson’s Disease Association,
Parkinson’s Disease Foundation, and the National Parkinson
Foundation. In January 2003, The Caldwell lab was selected as one
of only 11 worldwide to represent the research goals of The Michael
J. Fox Foundation for Parkinson’s Research in their Protein
Degradation Grant Initiative. For his combined teaching and
research efforts, Dr. Caldwell was also chosen as the recipient of
a 2003 CAREER award from the National Science Foundation, the most
prestigious honor for young faculty bestowed by that organization.
He is the author of 2 editions of a widely adopted textbook in
biotechnology sold worldwide in 3 languages by Harcourt. He
currently teaches courses in Molecular Genomics, Neuronal Signaling
Mechanisms, General Biology, and an acclaimed seminar on the
societal impact of the Human Genome Project.

Shelli N. Williams, B.Sc., is a doctoral candidate in the
Department of Biological Sciences at The University of Alabama in
Tuscaloosa, where she has attended the university as an
undergraduate and graduate student since 1997. Following her
early graduation magna cum laude from the university, Ms.
Williams began her graduate work in the laboratory of Dr. Guy A.
Caldwell. She has experience teaching introductory biology
courses to both major and non-major students and has served as
teaching assistant for a senior level discovery-based genomics
course funded by the Howard Hughes Medical Institute.

Kim A. Caldwell, Ph.D. is an Adjunct Assistant Professor
in the Department of Biological Sciences at The University of
Alabama Dr. Caldwell serves as an administrative liaison for a 1.8
million dollar grant from the Howard Hughes Medical Institute to
the Department of Biological Sciences at Alabama and is Director of
the HHMI Rural Science Scholars program at Alabama. She
has designed and taught courses in General Biology, a seminar on
the societal impact of the Human Genome Project, and course is a
entitled ‘The Language of Research’ which she teaches
jointly for Howard Hughes Research Interns at both Stillman College
and The University of Alabama.