Question

1. The E. coli LacZ will be used as a reporter gene and the expression of the LacZ gene product will be detected by the enzymatic cleavage of the compound X-gal producing blue staining within the embryo. Why was an E. coli LacZ gene used as a reporter gene?

2. Why is there a Drosophila weak basal promoter located on the P[lacZ] enhancer-trap element?

3. The E. coli LacZ will be used as a reporter gene and the expression of the LacZ gene product will be detected by the enzymatic cleavage of the compound X-gal producing blue staining within the embryo. If blue staining is observed, what does that indicate about the P[lacZ] enhancer-trap element within the Drosophila embryo?
4. What classes of genes will be studied in this experiment?
5. When you examine your prepared slides you observe that some embryos do not show a staining pattern, give a technical reason for this observation. Give a biological reason for this observation.

Answer 1

Answer-

• The lacZ was used as a reporter gene because this allows visual selection of transformants which can be easily analysed to identify the recombinants i.e the cells with our gene of interest. Also, this method is single step as compared to antibiotic selection i.e two-step therefore, is fast and easy.
• A weak basal promoter is used with P[lacZ] enhancer trap element to ensure that the reporter gene is expressed only when in proximity with a enhancer. This helps to study control of enhancer on gene expression and location of enhancer.
• This indicates the reporter gene is inserted in closeness to a enhancer, then only expression of lacZ is possible producing blue colonies.
• This can be used to identify genes that work at multiple developmental stages because gene expression is analysed by expression of reporter gene.
• Technical reason for no staining pattern on plate may be concentration of antibiotics is high that inhibited the growth of cells or ligation was not proper. Biological reason for no colonies would be gene is not inserted close to a enhancer.