Question

II. Transformation of E. coli with Plasmid DNA (PGLO) 1. Three LB (Luria Broth) agar plates are obtained. The plates contain: • Plate A: LB-Agar/Ampicillin/Arabinose • Plate B: LB-Agar/Ampicillin • Plate C: LB-Agar 2. Three microfuge tubes, each containing 60 uL of E. coli competent cells, are placed on ice. Competent cells are E. coli cells pre-treated with CaCl2, which makes their membranes perforated and able to take up DNA. 3. The following is added to the three tubes according to the figure below. The tubes are tapped to mix and incubated on ice for 15 minutes: Tube A: competent E. coli + PGLO plasmid DNA Tube B: competent E. coli + water Tube C: competent E. coli + water 4. The tubes are then transferred to a 42°C water bath and heat-shocked for 90 seconds. 5. The contents of each tube are then plated on their respective plates (content of tube A to plate A, and so on). Bacteria are spread on each plate, using a different disposable spreader each time. 6. Plates A, B, and C are incubated at 37°C overnight, and then transferred to the refrigerator. 7. The plates are examined the next day for growth and are also placed on a UV light box


1. Fill in the table above with what you observe on your plates.

2. Bacterial transformation occurred on which agar plate (s)? What evidence do you have that the bacteria were transformed here?

3. Which plates have glowing growth? Explain what causes bacteria to glow.

Answer 1

1)

	plate contents:LB/agar+....
plate	Ampicillin	arabinose	pGLO plasmid	water	growth	green glow
A	Yes	Yes	Yes	No	yes green coloured colonies	yes
B	Yes	NO	No	yes	no growth	No
C	No	No	No	yes	growth in the form of smear	No

Ans2)

As shown in ques fig Bacterial transformation occurred on agar plate A ,.

Growth on plate A is due to transformation because here bacterial colony growth occur in presence of antibiotic ampicillin/arabinose. normal E.coli are sensitive to ampicillin but E.coli-plasmidpGLO are resistant to ampicillinand also transformed bacteria acquire GFP from pGLO which produce green colour by using arabinase as substrate

so bacteria which grow on plate A are transformed .

Ans3)plate A have glowing growth because here transformed E. coli pGLO growing on LB/amp/arabinose will glow green. Because transformed E. coli have GFP which fluorescence green light using arbinose as a substrate. Arabinose: Induces expression of GFP by binding to the protein AraC. Arabinose creates a differential medium, which means that bothpGLO and non-pGLO cells can grow, but they look different (only the pGLO cells become fluorescent).While Ecoli pGLO does not give green colour when grow on LB plate because of absence of arabinose in media.