Question

Suppose that you carried out a Bacterial transformation of E. coli HA101 with pGLO plasmid experiment in the lab. During the experiment, plates with bacteria were inoculated from +GLO and -GLO microfuge tubes (LB (-) plate, LB/amp (-) plate, LB/amp (+) plate, and LB/amp/ara (+) plate).

            5) What does LB (-) plate contain?

6) What does LB/ amp (-) plate contain?

7) What does LB/amp (+) plate contain?

8) What does LB/amp/ara (+) plate contain?

Answer 1

ANSWER :-

The pGLO is a plasmid which has two important reporter genes in which one of the genes encodes for Green Fluorescent Protein (GFP) and the other encodes for antibiotic resistance(ampicillin). The gene for antibiotic resistance will allow the cells to resist the antibiotic Ampicillin and hence the cells will grow and reproduce. The GFP protein will be expressed only in the presence of a substrate called as Arabinose. So, only when transformation of both the genes occurs in the E.coli cells, there will be survival of the cells although their detection will require specific substrates such as addition of ampicillin to detect cells having ampicillin resistance gene and addition of arabinose to detect fluorescense indicating transformation has occured. LB indicates the Luria-Bertani media (Lysogenic broth) which is essential as a nutritional content for the growth of the microbial colonies.

1. LB(-) plate indicates that only LB media is added and there is no addition of any substrate such as ampicillin and arabinose. As there is no presence of pGLO plasmid indicated by 'minus', the cells will grow and will cover up the entire surface taking into consideration that the E.coli cells are not transformed and are not provided with any external stress inducing chemicals like ampicillin. So, a uniform growth can be seen in this case.
2. LB/amp (-) plate indicates that the plasmid pGLO is not added although the media is provided with the antibiotic ampicillin. This is the most important point you should understand. When ampicillin is present, the cells of bacteria will lack the gene from the plasmid which will provide resistance to the cells against ampicillin and hence the bacteria would not be able to grow and multiply forming colonies. So, there would be no colonies seen in this plate.
3. LB/amp(+) plate indicates that the plasmid is provided into the media along with the antibiotic ampicillin. As the cells can undergo the process of transformation, there will be resistance seen against ampicillin in the media and thus the transformed cells will definetely grow and form colonies which can be seen in the form of small circular structures on the media.
4. LB/amp/ara(+) plate will show presence of both the substrates namely arabinose and ampicillin in the media. The pGLO is also added and hence the transformants would be able to grow on the media, and due to the presence of gene for metabolizing arabinose, there would be expression of fluorescense which can be detected. So, the plate will show the transformed bacterial cells along with fluorescense.