Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 740

14.6. You wish to develop a genetically engineered E. coli to produce a particular known
protein. The protein converts a colorless substrate into a blue product. You have access
to a high-copy-number plasmid with a penicillin-resistant gene and normal reagents for
genetic engineering. Describe how you would engineer E. coli to produce this protein.
Consider: source of donor DNA; regulatory elements that need to be included; how the
donor and vector DNA are combined; how the vector DNA is inserted; and how you
would select for a genetically engineered cell to use in production.
14.7. You wish to express a particular peptide in E. coli using a high-copy-number plasmid.
You have the amino acid sequence for the peptide.
a. Explain the experimental process for generating and selecting the genetically
engineered E. coli using restriction enzymes, ligase, E. coli , plasmid with neomycin
resistance, and the known amino acid sequence.
b. What control elements would you place on the plasmid to regulate expression and to
prevent read-through?
14.8. a. There are three primary methods for obtaining donor DNAwhen performing genetic
engineering. Briefly describe those methods.
b. You need to produce a protein from humans in E. coli . You do not know the primary
amino acid sequence. You suspect that introns are present. Which method will you
use to obtain the donor DNA?
14.9. What is the difference between “transduction” and “transformation” when discussing
genes transfer to bacteria?