3. You wish to examine the regulation of the trp operon and engineer the followi

ID: 202895 • Letter: 3

Question

3. You wish to examine the regulation of the trp operon and engineer the following construct for gene expression in bacteria. In this case, luciferase acts as a reporter gene trpP trpo trp leader peptide luciferase A. In a normal, wild-type (WT) E. coli cell, would this engineered operon be considered positive inducible, positive repressible, negative inducible, or negative repressible, or some combination of these descriptions? B. To determine the effect of the leader peptide, you make two mutants of the reporter construct above. The first has two point mutations changing both of the TGG sequences to TGCs (G30C/G33C), and the last is lacking a leader peptide altogether (Neader). You transform these two constructs and a WT control into WT E. coli to measure gene expression using luciferase activity. Fill in the chart below to describe nutrient conditions that promote high luciferase expression. Nutrient Conditions Explanati to Promote Highest Luciferase Expression Construct WT-luciferase G30C/G33C- luciferase leader C. Just to make things more complicated, you 100 80 80 40 generate two mutants of the lac repressor and the trp repressor by swapping the DNA binding domains and the regulator binding domains: tce binding domain with/ggl DNA binding domain (TB-LDNA) and lac/ binding20 domain with DNA binding domain (LB- TDNA). In both cases, expression of these chimeric repressors is driven by constitutive promoters. However, your labels wear off, and you're not sure which tube is which. You transform one of the constructs into both WT E. coli and E. coli lacking the trp gene (trpR) Both strains of E. coli have the luciferase reporter gene that does not contain a leader peptide (Eleader) so that you can measure gene expression. You obtain the data to the right. Which construct did you transform: TB-LDNA or LB-TDNA? Explain how you know WT trpR high trp high lac low trp low lac high trp low lac low trp high lac

Explanation / Answer

a. In a normal wild-type E. coli cell, this engineered operon is considered as a negative repressible system. In the presence of higher concentration of tryptophan, the repressor protein (trpR) dimer form a complex with tryptophan, which block the operator site, as a result, RNA polymerase cannot transcribe the luciferase gene, thus no signal should be found. In the presence of lower concentration of tryptophan, operator site is free from the repressor protein, thus RNA polymerase can transcribe the luciferase gene, so that the signal should be observed. In this case, tryptophan repress the transcription of the trp operon genes, so this operon is called repressible operon. But this operon enter into the “off” state due to the active repressor protein, therefore, this operon is called negative repressible operon.

Construct

Nutrient Conditions to promote highest luciferase expression

Explanation

Wild-type-luciferase

Luciferase is expressed

The nutrient condition has lower tryptophan concentration, which induce transcription of luciferase.

G30C/G33C

Luciferase

It is not mentioned where these two G is located, in which region of trp operon or in the luciferase

leader

Increased luciferase expression compared to the wild-type construct.

The RNA segment 1, RNA segment 2 and RNA segment 3 of trpL alternately decreases. Leader peptide and attenuator sequence provides 10% of repression, which is released due to the deletion of leader peptide sequence.

c. TB-LDNA

In case of TB-LDNA transformation-lac DNA binding domain binds to the operator site, and no lac binding domain is present. But tryptophan binding domain is present.

high trp and high lac: tryptophan binds to the TB-no change in LDNA occurs, thus no expression of luciferase in wild-type as well as trpR mutant cells

Low trp and low lac: in the absence of trp, TB-LDNA promotes expression of luciferase in both wild type and trpR mutant cells.