Difference between revisions of "Part:BBa J10050"
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==Lambert_GA 2019 Characterization==
 
==Lambert_GA 2019 Characterization==
 
Lambert_GA 2019 tested several combinations of constitutive promoters and ribosomal binding sites to characterize each by measuring enzyme activity and therefore protein expression. The gene expressed, LacZ, codes for β-galactosidase (β-gal), which typically breaks down lactose. Instead of using lactose, we added the sugar ONPG (Ortho-Nitrophenyl-β-galactoside). β-gal breaks ONPG down into galactose and ONP (Ortho-Nitrophenol), which has a yellow color. If there is more ONP present, there is more enzymatic activity and therefore more expression of LacZ. We used a plate reader to measure absorbance at 420 nm, measuring yellow color, and 600nm, measuring cell density. We inputted those absorbance values into the Miller unit formula to calculate enzymatic activity per cell per milliliter.
 
Lambert_GA 2019 tested several combinations of constitutive promoters and ribosomal binding sites to characterize each by measuring enzyme activity and therefore protein expression. The gene expressed, LacZ, codes for β-galactosidase (β-gal), which typically breaks down lactose. Instead of using lactose, we added the sugar ONPG (Ortho-Nitrophenyl-β-galactoside). β-gal breaks ONPG down into galactose and ONP (Ortho-Nitrophenol), which has a yellow color. If there is more ONP present, there is more enzymatic activity and therefore more expression of LacZ. We used a plate reader to measure absorbance at 420 nm, measuring yellow color, and 600nm, measuring cell density. We inputted those absorbance values into the Miller unit formula to calculate enzymatic activity per cell per milliliter.
 +
<table>
 +
  <tr>
 +
    <th>Strain Identification Number</th>
 +
    <th>Promoter Part Number</th>
 +
    <th>RBS Part Number</th>
 +
    <th>Relative Strength of Promoter/RBS</th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
  <tr>
 +
    <th>Company</th>
 +
    <th>Contact</th>
 +
    <th>Country</th>
 +
    <th>      </th>
 +
  </tr>
 +
</table>
  
 
[[File:T--Lambert GA--tuningalldata.png|800px]]
 
[[File:T--Lambert GA--tuningalldata.png|800px]]

Revision as of 16:31, 21 October 2019


weak promoter + weak RBS + lacZa.GFP fusion

Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Weak constitutive promoter, weak RBS and the lacZa.GFP fusion protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 941

Usage and Biology

Lambert_GA 2019 Characterization

Lambert_GA 2019 tested several combinations of constitutive promoters and ribosomal binding sites to characterize each by measuring enzyme activity and therefore protein expression. The gene expressed, LacZ, codes for β-galactosidase (β-gal), which typically breaks down lactose. Instead of using lactose, we added the sugar ONPG (Ortho-Nitrophenyl-β-galactoside). β-gal breaks ONPG down into galactose and ONP (Ortho-Nitrophenol), which has a yellow color. If there is more ONP present, there is more enzymatic activity and therefore more expression of LacZ. We used a plate reader to measure absorbance at 420 nm, measuring yellow color, and 600nm, measuring cell density. We inputted those absorbance values into the Miller unit formula to calculate enzymatic activity per cell per milliliter.

Strain Identification Number Promoter Part Number RBS Part Number Relative Strength of Promoter/RBS
Company Contact Country
Company Contact Country
Company Contact Country
Company Contact Country
Company Contact Country
Company Contact Country
Company Contact Country
Company Contact Country
Company Contact Country
Company Contact Country

T--Lambert GA--tuningalldata.png