crb824: Molecular Technologies II
(industry-sited, hands-on laboratory course)
Year 1, Spring Semester (3 credits)
This second laboratory-intensive course explores biotechnologies with research, industrial and diagnostic applications—with a special emphasis on nucleic acid and protein analysis in diverse areas of biotechnology.
Classroom and lab activities include gene cloning, protein expression and purification on a manufacturing scale; the detection of genetically modified organisms and its implications for science and society; automation in biotechnology, and human genetic identity and its role in forensics.
You’ll also hear guest lectures on directed evolution through protein engineering and DNA, protein and cell microarrays.
The biomanufacturing laboratory component highlights many of the topics covered in Biotechnology Operations.
Session Topics from Spring 2021
Session 1: An introduction to Gene Cloning, Protein Expression, and Protein Purification; first laboratory exercises; Genetically modified organisms (GMOs) detection; Science, business, and regulatory impacts of GMOs in the agricultural biotechnology field.
Session 2: GMO detection continued; Protein-based method, ELISA (Enzyme Linked Immuno Sorbent Assay) and immunoassay strip test used. Purify plasmid DNA from the mini cultures of bacteria and then analyze the plasmid DNA with restriction enzymes to verify the correct target gene insert is present; GMO PCR reaction used.
Session 3: Inoculate and induce a fermentation culture of bacteria containing the target protein (zbFGF) using fermentors; Perform optimization, and batch record generation; Instruction on using of BioFlo fermentors. Process induced bacterial culture by centrifuging and will prepare a crude cell lysate, which will be subsequently purified in Sessions 4 and 5 and analyzed in Session 5; Batch record generation and product specifications will continue; Prepare solutions for chromatography downstream purification.
Session 4: Cation exchange chromatography on the crude bacterial lysate generated in Session 3b, using a Bio-Rad DuoFlow FPLC (Fast Performance Liquid Chromatography) system; Introduction to concepts surrounding genetic identity. Ppurify their own genomic DNA from cheek cells (buccal swabs); DNA will then be tested for genotyping their own samples; Analyze samples collected in a mock criminal case as well by purifying “touch DNA” using an automated magnetic silica purification method.
Session 5: Final formulation step of the target protein using tangential flow filtration or dialysis; Vialing to store the purified zbFGF product; Analyze and interpret the results of the STR genotyping reactions and discuss allele frequencies and the probability of specific genotypes and the power of discrimination or exclusion. Perform final analysis of the purified target zb-FGF protein; protein concentration determined using a commercially available colorimetric protein assay (BCA), while the purity and integrity of the final purified target protein will be determined using Experion chip analysis; Guest lecture on the science and business of genetic identity analysis.
Session 6: Tour Exact Sciences manufacturing/testing site and the Feynman Center (Promega GMP Biomanufacturing Facility). Deliver poster presentations with partners on their assigned topics.
Session 7: Introduction to laboratory automation and its role in biotechnology; Guest lecture on “Directed Evolution” (protein engineering) and role it plays in biotechnology. Tour and view demonstrations of various automated platforms in the R&D Integrated Systems laboratory at Promega Corporation. “Epigenetics/Epigenomics” introduced and significance in biotechnology discussed.