Question

Your friend decides to place Green Fluorescent Protein (GFP) under the control of the promoter from the lacZ gene we discussed in class. She put this expression plasmid into a bacterium. The promoter from the lacZ gene is diagrammed to the right.?

2) Your friend decides to place Green Fluorescent Protein (GFP) under the control of the promoter from the lacZ gene we discussed in class. She put this expression plasmid into a bacterium. The promoter from the lacZ gene is diagrammed to the right. RNA- CAP- polymerase start site for RNA binding binding site site (promoter) G) Recall that two important gene regulatory proteins that bind to this promoter are the lac repressor and the CAP protein. Your friend grows the bacteria with the expression plasmid on media (food) with no glucose and no lactose and found that the sleep-induced protein could not be purified from the bacteria. Is this surprising to you, given how the lacz promoter works? Explain how the binding of the gene regulatory factors lead to this situation. (5 points) operator Lacz gene b) How would you recommend your friend grow her bacteria so that she will get maximal expression cf GFP when using the lacZ promoter? (3 points) You decide to express GFP in a fly instead of a bacterium. The fly promoter has overlapping binding sites for Krüppel and Bicoid so that only one of the two proteins can bind to DNA at any one time (in other words, Krüppel and Bicoid cannot bind simultaneously). You conduct some experiments using a cell free transcriptional system, where you add the expression plasmid and equal quanti ties of Krüppel and Bicoid proteins and get the results in the Table 1 to the left. Table 1 in added GFP mRNA produced? none no Bicoid Krüppel and Bicoid no c) According to this data, which protein(s) is likely a gene activator? (2 points) d) According to this data, which prote in do you think has the stronger affinity for DNA? (2 points) e) Both Krüppel and Bicoid are sequence-specific binding proteins. Circle the portion(s) of the DNA rnolecule that these proteins would interact with for this property. (2 points) histones nitrogenous bases sugar am ino acids ADP dideoxyribose

Answer 1

2.a. It is not at all surprising. since the protein is inserted in the lac operon, its expression requires lactose or allolactose. in the absence of lactose, lac repressor binds to the operator site of the operon and prevents the binding of RNA polymerase, thereby inhibiting its transcription and expression.

this repression is relieved by lactose. lactose or allolactose binds to the repressor and would not allow the repressor to bind to operator region.

b. Usually, in molecular biology experiments, for protein expression under lac promoter, we use IPTG (Isopropyl beta D-1-thiogalactopyranoside) as an inducer. IPTG acts as a mimic of allolactose. therefore, the addition of IPTG activates the transcription of the protein under lac promoter.

c. according to the given data, bicoid is the gene activator because, when bicoid is added, GFP mRNA is produced.

d. Kruppel has a stronger affinity to DNA.

from the data given, we know that Bicoid is an activator and Kruppel is an inhibitor. however, when both the Bicoid and Kruppel are added, mRNA was not produced. it indicates the inhibitor has more affinity to DNA. the inhibitor is Kruppel.

e. these proteins may be interacting with the nitrogenous bases.