For the past two days in Biology, our teacher had us do a lab. We had to take bacteria (E-Coli, don't worry it was not the bacteria that will get us sick). Anyway we needed to follow a procedure to grow our own bacteria. It was quite interesting. First I will talk about the steps and then I will show you pictures of our results. Once I finish that I will show you the questions we were asked and how we answered them based on what we reserved.
Transformation Lab Guide:
Step 1: Label one closed micro test tube +pGLO and another -pGLO. Label both test tubes with your group's name. Place them in the foam tube rack.
Step 2: Open the tubes and using a sterile transfer pipet, transfer 250 ul of transformation solution.
Step 3: Place the tubes on ice.
Step 4: Use a sterile loop to pick up a single colony of bacteria from our 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.
Step 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. Why not?
Step 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.
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Step 7: While the tubes are sitting on ice, label your four agar plates on the bottom ( not the lid) as follows: Label the LB/amp plate: +pGLO; Label the LB/amp/ are plate: +pGLO; Label the other LB/ amp plate: -pGLO; Label the LB plate: -pGLO.
Step 8: Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 degrees Celsius, for exactly 50 seconds. Make sure to push the tubes all the way down the ram so the bottom of the tubes stick out and make contact with the water. When the 50 seconds are done, place both tubes on ice. For the best transformation results, the change from ice to 45 degrees Celsius and then back to the ice must be rapid. Incubate the tubes on ice for 2 minutes.
Step 9: Remove the rack containing the tube from the ice and place on bench top. Open a tube and, using a new sterile pipet, add 250 ul of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.
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Step 10 : Tap the closed tubes with your finger to mix. Using a sterile pipet for each tube, pipet 100 ul of the transformation and control suspensions onto the appropriate plates.
Step 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.
Step 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 degrees celsius incubator until the next day.
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Man that is a lot of directions. Now below I will show you our results and questions we needed to answer based on our observations.
This picture shows our LB positive plate. There wasn't any bacteria but it was streaky and smudged.
This was our LB/ amp negative plate. It was very empty and had no bacteria what so ever.
This was our LB/amp/ara positive plate. This plate had a lot (LOT) of bacteria growing on it. Under the UV light it also glows due to the jelly fish DNA.
In this picture the plate is labeled LB/amp positive. The yellow glowing dots are bacteria. They glow under the UV light also due to the jelly fish DNA.
Now that you have seen my groups results, I will show you are predictions we had to make on the plates before we got to see them. I put the questions red and our answers green.
Prediction Questions:
1) On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observers? Explain your predictions.
In Plate LB you would expect to find bacteria like the original.
2) If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions.
They would most likely be on Plate LB/amp because they have been transformed. Same with Plate LB/amp/ara.
3) Which plates should be compared to determine if any genetic transformation has occurred? Why?
LB v.s LB/amp because everything will grow on LB but different things will be on the LB/amp.
4) What is meant by a control plate? What purpose does a control serve?
LB is a good control because we didn't add or take away anything.
Now I will show you our Data collection questions. The questions will be in blue and our answers in green.
1) How much bacterial growth do you see on each plate, relatively speaking?
Either a lot of bacterial growth or none at all.
2) What color are the bacteria?
The bacteria colonies are a bright yellow color.
3) How many Bacterial colonies are on each plate?
There are a lot (LOT) of bacterial colonies, really too many to be able to count.
4) Which of the traits that you originally observed for E. coli did not seem to become altered? In the space below list these untransformed traits.
Original Trait Analysis of Observations
Yellow Blobs Still yellow blobs
5) Of the E. coli traits you originally noted, which seem now to be significantly different after preforming the transformation procedure? List those traits below and describe the changes that you observed.
New Trait Observed Change
New yellow blobs Blue tint glows under
glow blue tint. UV light.
6) If the genetically transformed cells have acquired the ability to live in the presence of those antibiotic ampicillin, then what might be inferred about the other genes on the plasmid that you used in your transformation procedure?
We added the arabinose to the plasmid it essentially created the GFP (free florescent protein).
7) From the results that you obtained, how could you prove that the changes that occurred were due to the procedure that you preformed?
Before we did the procedure the bacteria was normal. Afterwards it went through a transformation when it obtained a blue tinted glow due to obtaining a green florescent protein.
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