Question

1. Lab Review:

A) Explain why– control E. coli cells cannot grow on LBA+amp and LBA+amp+X-Gal.

B) Name a procedure which can replace the heat shock process and achieve the same goal:

C) If you used the pBLU plasmid as a vector to carry a desirable human gene into E. coli recipient cells. The insertion site is inside the β-galactosidase gene on the p-BLU plasmid. After the transformation, you plate the cells on LBA+amp+X-Gal to screen for E. coli cells with the desirable gene. You saw both blue and white colonies on the plate. Which type of colonies carry the desirable human gene?
A. White        B. Blue             C. both             D. neither one

Explain WHY?

Answer 1

A) The Control group of E.coli bacterial cells does not contain the plasmid DNA which provides antibiotic-resistant in the selection medium containing ampicillin therefore, the control group can not grow in the antibiotic selection medium.

B) Another procedure that can be used in place of the heat shock process which called electroporation method in which competent cells and plasmid placed into electroporation cells and under the electric field bacterial cell wall allows to enter plasmid.

C) Option A is the right answer in which the negative selection or loss of function of the marker gene explained which colony has the gene of interest. In this process gene of interest inserted into the beta-galactosidase gene and disrupt its function so if the gene is properly inserted into the frame beta-galactosidase then that enzyme becomes inactive and can not catalyze the X-gal reaction therefore that colonies have white appearance.