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.
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