Exercise 6.3 Explain what happening (phenotype) to the following E. coli mutants when they are grown in two different media, which are glucose only and lactose only. Give your reasoning for your answer. (a) Strain with a mutation in the gene encoding the lac repressor; the mutant repressor cannot bind allolactose. (2 Marks) (b) Strain with a mutation in the gene encoding CAP; the mutant form of CAP binds but cannot release cAMP. (8 Marks)

The following polynucleotide was synthesized and used as a template forpeptide synthesis in a cell-free system from E. coli.                                    …AUAUAUAUAUAUAU… If the same polynucleotide described is used with a mitochondriaderivedcell-free protein-synthesizing system, the product is                                  …Met–Tyr–Met–Tyr–Met–Tyr…What does this say about differences between the mitochondrial and bacterial codes?

A Leu →Ala mutation at a site buried in the core of the enzyme lysozymeis found to be destabilizing. Explain the observed effect of this mutationon lysozyme stability by predicting how enthalpy (ΔH°), conformationalentropy (ΔS°peptide), and the hydrophobic effect (ΔS°solvent) are expected to change for the mutant compared to wild-type lysozyme. Explain how ΔG°for unfolding is affected by your predicted changes in enthalpy or entropy.

* REQUIRED 34. The diagram below represents one of a number of different types of mutations that can occur in DNA ....CACTAGCAG.... DNA Sequence mutation ....CACTAACAG.... DNA Sequence This mutation can best be described as the the insertion of an adenine (A) base into both strands of the DNA molecule pairing of an adenine (A) base with thymine (T) the substitution of an adenine (A) base for guanine (G) deletion of an adenine (A) base from the DNA molecule 0 1

. (a) Explain the biochemical basis for the fact that one can synchro- nize cell populations þy treating them with deoxythymidine. (b) Explain the apparent paradox that dATP at low concentrations is an activator of ribonucleotide reductase, whereas at higher concen- trations it becomes inhibitory.

Clary Fray used the pET vector system to express her prokaryotic amylase enzyme. She added peptone into her culture broth of BL21(DE3) Escherichia coli strain to induce protein expression. At the end of the experiment, she discovered that her protein was not expressed. She repeated three more times but her protein of interest was still not produced. (i) (ii) (iii) (iv) (v) Explain the reason why Clary failed to obtain her protein of interest and suggest a solution to troubleshoot this problem. Clary plans to express her protein along with a polyhistidine-tag. Explain the importance of His-tag in protein work. Is DH5a Escherichia coli suitable to propagate the plasmid before protein expression? Besides heat shock method, elaborate another procedure Clary could utilize to transform the recombinant pET vector into the host cell. If her supervisor instructs her to express a gene from gold fish (Carassius auratus), is the expression system she is using now suitable for this experiment?…

There are six classes of enzymes, namely oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. (i) (ii) Cell disruption is one of the steps performed during enzyme production. Explain the reason of performing cell disruption in enzyme production and provide THREE (3) enzyme extraction methods for this type of enzyme. Explain the principles of gel electrophoresis as one of the methods used for enzyme purification.

Wild-type E. coli cells can synthesize all 20 common amino acids, but some mutants, called amino acid auxotrophs, are unable to synthesize a specific amino acid and require its addition to the culture medium for optimal growth. Besides their role in protein synthesis, some amino acids are also precursors for other nitrogenous cell products. Consider the three amino acid auxotrophs that are unable to synthesize glycine, glutamine, and aspartate, respectively. Match each nitrogenous product with the mutant or mutants that would fail to synthesize it. Each mutant may fail to synthesize more than one nitrogenous product, and some nitrogenous products may fail to be synthesized by more than one mutant. glycine auxotrophs glutamine auxotrophs aspartate auxotrophs Answer Bank adenine nucleotides cytosine nucleotides guanine nucleotides uridine nucleotides

Consider the biochemical pathway shown here. Suppose that a strain of bacteria must synthesize compound 4 to survive and divide. Successful survival and division of bacteria is observed as growth of colonies on an agar plate. This strain of bacteria can grow colonies on minimal medium as long as it is supplemented with compound 1. You are in a lab that has isolated several mutants of this strain. You find that these mutants cannot grow on minimal medium supplemented with compound 1, though they can grow colonies if supplemented with compound 4. Considering what you know about the Beadle-Tatum experiments, which of the following statements would be one that should be true?

The purpose of the progress curve is to determine if an enzymatic reaction rate remains constant for given concentrations of enzyme and substrate, for a given assay time (20 min). How long did the rate of reaction of WGAP remain constant in your experiments? How do you know? What would cause the reaction rate to not be constant and plateau after some time?