Biotechnology Applications and Bioprocessing: From Molecule to Product
Core concepts
Trace a product from construct to release. Recombinant DNA work uses restriction enzymes (palindromic cut sites producing sticky or blunt ends), ligase, vectors with origin of replication, selectable marker, and promoter, and transformation methods (heat shock, electroporation, transduction). PCR needs template, primers, dNTPs, polymerase, and buffer, and cycles through denaturation, annealing, and extension; real-time PCR quantifies. Sequencing (Sanger, next-generation) and CRISPR editing are core tools. Expression systems differ: E. coli is fast and cheap but lacks glycosylation; yeast and mammalian cells add post-translational modification at higher cost. Bioprocessing covers upstream (inoculum, media, bioreactor control of pH, temperature, dissolved oxygen, agitation) and downstream (centrifugation, filtration, chromatography - ion exchange, size exclusion, affinity - and formulation). Know yield, titer, and purification fold calculations, and quality attributes such as sterility and endotoxin limits.
Traps
Do not assume E. coli can produce a fully glycosylated human protein. In chromatography, ion exchange separates by charge, size exclusion by size, affinity by specific binding. A high-yield step with low purity can still fail release criteria. Watch units: colony-forming units per mL versus cells per mL, and dilution factors in titer math.
Worked example
A 1000 L bioreactor yields 2 g/L product, so 2000 g total. If recovery is 80 percent and purification is 50 percent, final mass = 2000 × 0.8 × 0.5 = 800 g. Track each step multiplicatively; a common error is adding percentages instead of multiplying them.