Question

1. As a general principle, in order to validate results in scientific experiments, scientists use controls.  A control is a group that is designed to demonstrate that the results/changes observed in an experiment were because of the procedure you performed.  It sets a baseline value with which the scientist can compare with experimental sample and interpret results, as was demonstrated in the previous question.  In this question, we are asking you to describe the control experiments, using the same transformation protocol, with pGLO plasmid.  

1. You want to test whether the E. coli cells were viable (alive).  How would you test this?  Consider the following:  (a) do you add the plasmid to the cells? (b) what plate do you use – NA or NA-AMP and (c) do you use visible light or UV light?

2. You want to test if the resistance of cells to Ampicillin was produced only as a result of cells taking up pGLO.  

Answer 1

A). E. coli cells are viable or not can be checked by various stins like propidium iodide and SYTO9 dye which will stain live cells green and dead cells red colored by checking whether cell membrane is disturbed.

a). For detection of whether E. coli cells were viable or not I would add the pGLO plasmid in cells. This pGLO plasmids contains GFP(greng flourescense protein) encoding gene and ampicillin resistance gene as marker.

b). I will use Nutrient agar plate with amp(ampicillin) because only cells with pGLO plasmid will be able to grow due to presence of ampicillin resistance gene in plasmid of viable e.coli.

c). For the detection of flourescense by GFP protein can be detected under uv light only.

B.) To check whether resistance of cell is due to plasmid or it is already present in normal cell. So we will grow the cells on NA-amp( with arabinose) because the pGLO plasmid having bidirectional promoter which get activated in presence of arabinose and express GFP and ampicillin genes.

So only resistance cells will grow and if the cells present on the plates also gives flourescense under uv light it suggests that the resistance is provided by pGLO gene presence.we will also grow our original E. coli cells( which were used for pGLO plasmid insertion) on NA-amp( with arabinose),if cells doesn't grow it will confirm that the resistance is provided by pGLO plasmid and not by default present in cell.