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Thread: doubt

  1. #1 doubt 
    Forum Freshman
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    OK,

    The experiment consist to know where is the enzyme(B-lactamase) located( in the periplasm or cytoplasma) so, the bacteria is treated with osmotic shock, 3 steps: EDTA, hipertonic solution and the hipotonic solution. Before this, is necesary to centrifugate and finally I have a pellet and supernatant ( of each step) in wish add a indicator ( Nitrocefina).
    And my doubt is...what results I expect to see in each step?

    in where place I expect to see color of the indicator if is located in periplasm(or cytoplasm) in the 3 steps: in the pellet or supernatant?

    example: the enzyme is located in cytoplam

    steps 1, EDTA: supernatant (color or not color?)
    pellet (color or not color?)

    step2, hypertonic: supernatant(?)
    pellet(?)
    step3, hipotonic: supernatant (?)
    pellet(?)


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  3. #2  
    Forum Cosmic Wizard i_feel_tiredsleepy's Avatar
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    Ok in Step 1

    The EDTA is destroying the outer membrane, so if the lactamase was in the periplasm you would expect it in the supernatant. If it was in the cytoplasm it would be in the pellet.

    Step 2:

    If the osmotic pressure is sufficient to lyse the cell the indicator should be in the supernatant.

    Step 3:

    It should be in the pellet. Because the cell will shrivel and proteins shouldn't diffuse out of the cell and remain trapped. I think.


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  4. #3  
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    thanks!!

    you are so nice


    :wink:
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  5. #4  
    Forum Cosmic Wizard i_feel_tiredsleepy's Avatar
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    I must have been on crack when I answered this because I mixed up hyper and hypo tonic lol... I'm surprised no one caught this.
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