BioEngineering Lab Techniques: A Novel Lab Course for Protein Expression in Bacterial and Mammalian Cells

Jacob James Elmer; Andre Palmer; Jessica O Winter

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Conference: 2018 ASEE Annual Conference & Exposition
Location: Salt Lake City, Utah
Publication Date: June 23, 2018
Start Date: June 23, 2018
End Date: July 27, 2018
Conference Session: Novel Classrooms
Tagged Division: Chemical Engineering
Page Count: 18
DOI: 10.18260/1-2--29850
Permanent URL: https://peer.asee.org/29850
Download Count: 1563

Paper Authors

biography

Jacob James Elmer Villanova University

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Dr. Elmer earned dual B.S. degrees in Biology and Chemical Engineering from the University of Missouri Rolla in 2003 and obtained a PhD in Chemical Engineering from Ohio State University in 2007. After a short posdoc at Arizona State University and some adjunct teaching at Grand Canyon University, he secured an Assistant Professorship at Villanova University in the Chemical Engineering department. He currently teaches heat transfer and several biochemical engineering electives (Lab Techniques, Protein Engineering, etc.). His research focuses on developing novel blood substitutes and optimizing gene therapy treatments.

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Andre Palmer Ohio State University

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Jessica O Winter P.E. Ohio State University

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Abstract

BioEngineering Lab Techniques is an interdisciplinary lecture + lab course that is designed to give undergraduate students hands-on experience with cutting edge molecular biology and cell culture techniques that are utilized for recombinant protein production. Specifically, the students learn and perform every step necessary to express fluorescent proteins (e.g. GFP, mCherry) and chromoproteins (e.g. aeBlue, tsPurple, eforRed, et al.) in bacterial and animal cells by completing the following modules.

Module 1: Molecular Genetics & Cloning The course begins with an overview of DNA and different cloning techniques, including PCR and Circular Polymerase Extension Cloning (CPEC). The first module includes 5 labs, in which the students amplify the gene for a colorful chromoprotein with PCR (Lab 1), clone that gene into an expression plasmid (Lab 2), transform the new plasmid into E. coli (Lab 3), then extract (Lab 4) and sequence (Lab 5) the plasmid.

Module 2: Bacterial Fermentation – Expression of Chromoproteins In Module 2, the students use the expression plasmids from Module 1 to express their recombinant chromoproteins in E. coli (Lab 6), then purify the protein with either chitin affinity chromatography (Lab 7) or immobilized metal affinity chromatography (Lab 8). The purified protein is then characterized to determine its purity (PAGE), concentration (BCA assay), and absorbance spectrum in Lab 9, while Lab 10 is a virtual exercise that introduces the students to protein modeling software (FoldIt, SwissPDB).

Module 3: Animal Cell Culture – Genomic Integration of Fluorescent Proteins As an alternative to bacterial protein expression, Module 3 shows students how to integrate genes into mammalian cell chromosomes to create stable transgenic animal cell lines for protein expression. Specifically, students begin by learning to passage adherent cells (Lab 11), then they prepare plasmids for the genomic integration of an mCherry gene (Lab 12) and transfect those plasmids into animal cells (Lab 13). Transgene expression is detected with fluorescent microscopy (Lab 14) and the course concludes with a virtual demonstration of transfectant selection & expansion of the transformed cells in a WAVE bioreactor (Lab 15).

In summary, this course teaches students every step in recombinant protein production from start (plasmid construction) to finish (protein purification & characterization). Student feedback to surveys is highly positive and graduates indicate that the course helped them prepare for and acquire their current jobs.

Course materials are available upon request from the instructor.

Citation
Format

Elmer, J. J., & Palmer, A., & Winter, J. O. (2018, June), BioEngineering Lab Techniques: A Novel Lab Course for Protein Expression in Bacterial and Mammalian Cells Paper presented at 2018 ASEE Annual Conference & Exposition , Salt Lake City, Utah. 10.18260/1-2--29850

TY  - CPAPER
AB  - BioEngineering Lab Techniques is an interdisciplinary lecture + lab course that is designed to give undergraduate students hands-on experience with cutting edge molecular biology and cell culture techniques that are utilized for recombinant protein production.  Specifically, the students learn and perform every step necessary to express fluorescent proteins (e.g. GFP, mCherry) and chromoproteins (e.g. aeBlue, tsPurple, eforRed, et al.) in bacterial and animal cells by completing the following modules.

Module 1: Molecular Genetics &amp; Cloning
The course begins with an overview of DNA and different cloning techniques, including PCR and Circular Polymerase Extension Cloning (CPEC).  The first module includes 5 labs, in which the students amplify the gene for a colorful chromoprotein with PCR (Lab 1), clone that gene into an expression plasmid (Lab 2), transform the new plasmid into E. coli (Lab 3), then extract (Lab 4) and sequence (Lab 5) the plasmid.  

Module 2: Bacterial Fermentation – Expression of Chromoproteins
In Module 2, the students use the expression plasmids from Module 1 to express their recombinant chromoproteins in E. coli (Lab 6), then purify the protein with either chitin affinity chromatography (Lab 7) or immobilized metal affinity chromatography (Lab 8).  The purified protein is then characterized to determine its purity (PAGE), concentration (BCA assay), and absorbance spectrum in Lab 9, while Lab 10 is a virtual exercise that introduces the students to protein modeling software (FoldIt, SwissPDB). 

Module 3: Animal Cell Culture – Genomic Integration of Fluorescent Proteins
As an alternative to bacterial protein expression, Module 3 shows students how to integrate genes into mammalian cell chromosomes to create stable transgenic animal cell lines for protein expression.  Specifically, students begin by learning to passage adherent cells (Lab 11), then they prepare plasmids for the genomic integration of an mCherry gene (Lab 12) and transfect those plasmids into animal cells (Lab 13).  Transgene expression is detected with fluorescent microscopy (Lab 14) and the course concludes with a virtual demonstration of transfectant selection &amp; expansion of the transformed cells in a WAVE bioreactor (Lab 15).

In summary, this course teaches students every step in recombinant protein production from start (plasmid construction) to finish (protein purification &amp; characterization).  Student feedback to surveys is highly positive and graduates indicate that the course helped them prepare for and acquire their current jobs.  

Course materials are available upon request from the instructor.

AU  - Jacob James Elmer
AU  - Andre Palmer
AU  - Jessica O Winter P.E.
CY  - Salt Lake City, Utah
DA  - 2018/06/23
PB  - ASEE Conferences
TI  - BioEngineering Lab Techniques: A Novel Lab Course for Protein Expression in Bacterial and Mammalian Cells
UR  - https://peer.asee.org/29850
DO  - 10.18260/1-2--29850
ER  -