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pGLO Lab Report

Purpose
In this experiment, we will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP) and learn about the process of moving genes from one organism to another with the aid of a plasmid.

Materials
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E. coli starter plate 1 LB 2 LB/amp 1 LB/amp/ara Transformation Solution LB nutrient broth Inoculation loops Pipets Foam microcentrifuge tube holder/float Container (such as foam cup) full of crushed ice (not cubed ice) Marking pen Copy of Quick Guide Microcentrifuge tubes Rehydrated pGLO plasmid 42 degrees Celsius water bath and thermometer UV Light 37 degrees Celsius incubator 2-20 microliter adjustable volume micropipets 2-20 microliter micropipet tips

Poured agar plates

1 1 7 (1 pk of 10) 4 1 1 1 1 2 1 vial 1 1 1 1 1

Procedure
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Label one closed micro test tube + pGLO and another pGLO. Label both tubes with your groups name. Place them in the foam tube rack. Open the tubes. Using a sterile transfer pipet, transfer 250 microliters of transformation solution (CaC12) into each tube. Place the tubes on crushed ice. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pGLO tube. Immerse the loop into the transformation solution at the bottom of the +pGLO tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution. Check for floating chunks.

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Place the tube back in the tube rack in the ice. Using a new sterile loop, pick up a single colony of bacteria from your starter plate. Pick up the -pGLO tube. Immerse the loop into the transformation solution at the bottom of the -pGLO tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution. Check for floating chunks. Place the tube back in the tube rack in the ice. 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. Mix the loopful into the cell suspension of the +pGLO tube. Pipet 10 microliters of pGLO plasmid into the +pGLO tube. Mix the +pGLO tube. Close the pGLO tube. Return the pGLO tube to the rack on ice. Push the bues all the way down in the rack so the bottom of the tubes sticks out and makes contact with the ice. Incubate the tubes on ice for 10 minutes. Label the four agar plates on the bottom (not the lid). Label the first +pGLO, LB/amp. Label the second +pGLO, LB/amp/ara. Label the third -pGLO, LB/amp. Label the fourth -pGLO, LB. Set the water bath to 42 degrees Celsius. Using the foam rack as a holder, transfer both the +pGLO and pGLO tubes into the water bath. 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. Leave the tubes in the water bath for exactly fifty seconds. Immediately place both tubes back on ice. Incubate tubes on ice for two minutes. Remove the rack containing the tubes from the ice. Place the rack on the bench top. Open the +pGLO tube. Using a new sterile pipet, add 250 microliters of LB nutrient broth to the +pGLO tube. Reclose the +pGLO tube. Open the pGLO tube. Using a new sterile pipet, add 250 microliters of LB nutrient broth the the pGLO tube. Reclose the pGLO tube. Incubate both tubes for ten minutes at room temperature. Gently flick the closed bues with your finger to mix. Open the +pGLO tube.

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Remove the lid to the agar plate labeled +pGLO, LB/amp. Using a new sterile pipet, pipet 100 microliters onto the agar plate labeled +pGLO, LB/amp. Replace the lid to the agar plate labeled +pGLO, LB/amp. Remove the lid to the agar plate labeled +pGLO, LB/amp/ara. Using the same sterile pipet, pipet 100 microliters onto the agar plate labeled +pGLO, LB/amp/ara. Replace the lid to the agar plate labeled +pGLO, LB/amp/ara. Remove the lid to the agar plate labeled -pGLO, LB/amp. Using a new sterile pipet, pipet 100 microliters onto the agar plate labeled -pGLO, LB/amp. Replace the lid to the agar plate labeled -pGLO, LB/amp. Remove the lid to the agar plate labeled -pGLO, LB. Using the same sterile pipet, pipet 100 microliters onto the agar plate labeled pGLO, LB. Replace the lid to the agar plate labeled -pGLO, LB. Remove the lid to the agar plate labeled +pGLO, LB/amp/ara. Using a new sterile loop, spread the suspensions evenly around the surface of the agar. Skate the flat surface back and forth across the plate surface. Replace the lid to the agar plate labeled +pGLO, LB/amp/ara. Remove the lid to the agar plate labeled +pGLO, LB/amp. Using a new sterile loop, spread the suspensions evenly around the surface of the agar. Replace the lid to the agar plate labeled +pGLO, LB/amp. Remove the lid to the agar plate labeled -pGLO, LB/amp. Using a new sterile loop, spread the suspensions evenly around the surface of the agar. Replace the lid to the agar plate labeled -pGLO, LB/amp. Remove the lid to the agar plate labeled -pGLO, LB. Using a new sterile loop, spread the suspensions evenly around the surface of the agar. Replace the lid to the agar plate labeled -pGLO, LB. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack. Place the stack upside down in the 37 degrees Celsius incubator until the next day.

Hypothesis
Although my original hypothesis was misplaced, I recall thinking that:

-pGLO, LB -pGLO, LB/amp +pGLO, LB/amp

Bacteria Yes No Yes

Glowing Bacteria No No No

+pGLO, LB/amp/ara

Yes

Yes

Results

Bacteria -pGLO, LB -pGLO, LB/amp +pGLO, LB/amp +pGLO, LB/amp/ara Yes No Yes Yes

Glowing Bacteria No No No Yes

% Coverage of Bacteria 95% 0% 50% 80%

Conclusion
The purpose of this lab was to make bacteria glow and learn how to move genes between organisms using a plasmid. The controlled variables were the bacteria, the agar, and the temperature at which they incubated. Experimental variables were the pGLO plasmid, the arabinose, and the ampicillin. In our lab, the pGLO, LB nutrient agar plate grew bacteria that did not glow. The pGLO, LB/amp nutrient agar plate did not grow any bacteria. The +pGLO, LB/amp nutrient agar plate grew bacteria that did not glow. The +pGLO, LB/amp/ara nutrient agar plate grew bacteria that did glow. From these results, I conclude that on normal LB plates,

bacteria will grow. Also, that ampicillin will kill normal bacteria. In addition, bacteria that have the pGLO plasmid can survive ampicillin. This evidence also led me to conclude that the pGLO plasmid plus arabinose will cause bacteria to glow. This experiment should be repeated several more times to further prove and support these conclusions. My hypothesis was that bacteria would grow in all of the nutrient agar plates except for the pGLO, LB/amp nutrient agar plate. Also, the bacteria in the +pGLO, LB/amp/ara nutrient agar plate would glow. The first experiment proved my hypothesis exactly right. However, the experiment must be repeated several more times to be sure. During this pGLO experiment, I learned how to move plasmids containing DNA into an agar plate of bacteria. I also learned that some jellyfish glow, which I did not previously know. My preliminary theory is that, with the combination of LB nutrient agar, ampicillin, arabinose, and a pGLO plasmid, bacteria in a nutrient agar plate will grow. I theorize this because this is what the result of the experiment was. To further experiment with this and to support this conclusion, I would take another antibiotic, such as Amoxicillin, and add it to the pGLO plasmid. Then you could have up to ten nutrient agar plates to thoroughly test the tentative conlusion: -pGLO, LB; -pGLO, LB/amp; -pGLO, LB/amo; -pGLO, LB/amp/amo; +pGLO, LB/amp; +pGLO, LB/amo; +pGLO, LB/amp/amo; +pGLO, LB/amp/ara; +pGLO, LB/amo/ara; and +pGLO, LB/amp/amo/ara.