Homework 3

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Answer Key in PDF format

Bioc471a/571a Homework 2 - Due at the start of class September 11

1. (2 pts) What does it mean to "titer a phage stock"? If your objective is to purify as much phage DNA as possible, why does it matter how much phage stock you add to the bacterial liquid culture, doesn't it all just mix around and lead to 100% infection eventually?







2. (2 pts.) What two genetic strategies have been developed to increase the percent of phage particles containing recombinant molecules? Are these genetic selections or a genetic screens? Explain.







3. (2 pts.) Briefly describe the key components of the pET bacterial expression system. Using 35S-methionine labeling media (contains radioactive methionine as the source of this amino acid), it has been observed that within 4 hours of adding IPTG to the culture, over 90% of the newly synthesized proteins in cell extracts are derived from the insert coding sequence in the pET vector. What explains the dramatic decrease in the synthesis of E. coli proteins during the induction period?








4. (2 pts.) What are two explanations for a recombinant l genomic phage that repeatedly hybridizes to your probe with only a faint signal throughout the plaque purification process? What would explain >200 intense hybridizing phage plaques from a cDNA library screening using an uncharacterized probe?








5. (2 pts.) What is the advantage of using site directed mutagenesis rather than deletion analysis to study protein structure and function? Why is alanine a good amino acid to use for replacement in a protein coding sequence? What other amino acid would be a good choice for "scanning" based on its chemical structure?