Question

A) Imagine that you found a white colony on a LB plate (containing Ampicillin, X-gal, and arabinose) that did not fluoresce green E. coli. Propose a hypothesis for how this could have occured.

B) How could you test your hypothesis using techniques and equipment in a lab.

Answer 1

A) HYPOTHESIS:

-pGLO/LB plate (the control): Without the pGLO plasmid, the bacteria inside the plate will grow, since the bacteria are resistant to the antibiotics. However, the bacteria will not glow in the dark, since there is no pGLO plasmid inside the plate.

-pGLO/LB/amp plate: Inside this plate, the bacteria will neither grow nor glow, since there is antibiotic to stop the growth of the bacteria, and pGLO plasmid is not presented in the plate.

+pGLO/LB/amp plate: For this plate, the bacteria will grow yet not glow.

+pGLO/LB/amp/ara plate: As this plate has LB, amp, and arabinose (a type of sugar). The bacteria will both grow and glow.  

B)METHODOLOGY

MATERIALS:

1 E.Coli starter plate

4 agar plates (labeled as such):

·         +pGLO: LB/amp

·         +pGLO: LB/amp/ara

·         -pGLO: LB

·         -pGLO: LB/amp

4 microtubules:

·         1 microtubule containing transformation solution

·         1 microtubule containing LB broth

·         1 empty microtube labeled +

·         1 empty microtubule labeled -

A foam microtubule holder

1 container with ice water

1 package of sterile inoculation loops

4 sterile pipettes

1 micropipette (200 - 1000 ul) and micropipette tips

1 micropipette (20 - 200 ul) and micropipette tips

1 Sharpie

1 UV light-lamp

PROCEDURE

1.     Label one closed micro test tube +pGLO and another –pGLO. Label both tubes with your group’s name. Place them in the foam tube rack.

2.     Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution (CaC12).

3.     Place the tubes on ice.

4.     Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the -pGLO tube.

5.     Examine the pGLO plasmid DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on ice. Also close the –pGLO tube. Do not add plasmid DNA to the -pGLO tube.

6.     Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.

7.     While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid) as follows: Label one LB/amp plate: +pGLO; Label the LB/amp/ara plate:  +pGLO; Label the other LB/amp plate: -pGLO; Label the LB plate: -pGLO.

8.     Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 °C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the change from the ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2 minutes.

9.     Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 µl of LB nutrient broth to the tub and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.

10.  Tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet 100 µl of the transformation and control suspensions onto the appropriate plates.

11.  Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface.

12.  Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack upside down in the 37°C incubator until the next day.

RESULTS:

	+pGLO: LB/amp	+pGLO: LB/amp/ara	-pGLO: LB	-pGLO: LB/amp
Growth/GLOw	Did grew/ Did NOT glow.	Did grew/ Did glow.	Did grew/ Did NOT glow.	Did NOT grow/ Did NOT glow.
Why/Why Not?	The cells with the plasmid had ampicillin resistance; however, it did not have arabinose to enable the Operon system for glowing.	The cells had ampicillin resistance and had arabinose to enable the Operon system for glowing.	The cells without the plasmid did not have ampicillin resistance. Also, it did not have arabinose to enable the Operon system for glowing.	The cells did not have ampicillin resistance. Moreover, it did not have arabinose to enable the Operon system for glowing.

CONCLUSION:

As shown in the result, only the LB/ amp/ ara plate could glow in the dark. The control of this lab is the –pGLO: LB/amp plate where the bacteria would neither grow nor glow. The final result fails to reject my hypothesis. On the other hand, this lab also demonstrates the inducible operon system. Originally, the system contained an active repressor that stopped the production of GFP --- green fluorescent protein. Nevertheless, as arabinose (a type of sugar) was brought into the system from an outside source, the repressor became inactive. Arabinose enabled the growth of GFP (unlocked the operator). Thus, the bacteria would be able to glow in the dark. Yet, this process would not last forever, since the system would create the arabinase to digest the arabinose. As the arabinose was used up, there would be nothing left to inactivate the repressor; therefore, the bacteria would stop glowing in the dark. Two constants for this lab were temperature of the incubation and the amount of bacteria in the plates. Although the final result indicated that the lab was successful, there was still some source of errors. For example: 1) my group did not scratch the bacteria onto the right plate when transferring. 2) There was some false measurement when we measured the solution for 100ul and 200 ul, since the label was hard to read. 3) We accidently used the same pipette for different tubes and plates. We forgot to change our pipettes. This lab did not only demonstrate and explain why the bacteria would glow under certain conditions, but also combined all the knowledge we have learnt in this unit. Overall, this lab was successful.