Difference between revisions of "Team:Gifu/InterLab"

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&nbsp;&nbsp;We constructed 5 plasmids [positive control, negative control, device1~3] by using BioBricks and then transformed to <i>E. coli</i> K-12 JM109. We checked them by colony direct PCR and plasmids sequence. We cultivated correct bacterias by shaking until OD660:0.5. Cultivated mediums were　transferred to 96-well plate by 100mL. We read the absorbance.
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<p>&nbsp;&nbsp;We constructed 5 plasmids [positive control, negative control, device1~3] by using BioBricks and then transformed to <i>E. coli</i> K-12 JM109. We checked them by colony direct PCR and plasmids sequence. We cultivated correct bacterias by shaking until OD660:0.5. Cultivated mediums were　transferred to 96-well plate by 100mL. We read the absorbance.</p>
 
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&nbsp;&nbsp;Setting of plate reader is following:<br></font>
 
&nbsp;&nbsp;Setting of plate reader is following:<br></font>

Revision as of 04:00, 31 August 2015


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Inter Lab Study


Equipment

  • BioSHAKER TA-12R
    • use for shaking culture



  • Safire ART-NR:F129013
    • use for measurement of absorbance




Method of Measurement

      We constructed 5 plasmids [positive control, negative control, device1~3] by using BioBricks and then transformed to E. coli K-12 JM109. We checked them by colony direct PCR and plasmids sequence. We cultivated correct bacterias by shaking until OD660:0.5. Cultivated mediums were　transferred to 96-well plate by 100mL. We read the absorbance.


      Setting of plate reader is following:



Result

  • Definition
    • We expressed absorbance as relative value for　positive control.
    This expression is following:
      Sample : Absorbance in sample
      NCav : Average of absorbance in negative control
      PCav : Average of absorbance in positive control



  • Value
    • Relative absorbance values for positive control are shown below.


    FIG1 Relative absorbance values
    20A : Device1 (J23101 + I13504)
    22A : Device2 (J23106 + I13504)
    22K : Device3 (J23117 + I13504)




Discussion

      The value of device1 is much higher than that of device2 and 3. This is why each device is different from promoter sequence, especially -10 and -35 region.



    Promoter sequence in device1 resembles consensus sequence. On the other hand, device2 and 3 is not similar than device1. This is how each GFP expression is different.











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