View source for Part:BBa B0035


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<partinfo>BBa_B0035 short</partinfo>

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_B0035 SequenceAndFeatures</partinfo>

===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.
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<table style="width:100%">
 <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>
    <td>R (positive control)</td>
    <td>BBa_J23115</td>
    <td>BBa_B0035</td>
    <td>Reference/Reference</td>
  </tr>
   <tr>
    <td>1</td>
    <td>BBa_J23113</td>
    <td>BBa_B0031</td>
    <td>Weak/Weak</td>
  </tr>
   <tr>
    <td>2</td>
    <td>BBa_J23113</td>
    <td>BBa_B0032</td>
    <td>Weak/Medium</td>
  </tr>
   <tr>
    <td>3</td>
    <td>BBa_J23113</td>
    <td>BBa_B0034</td>
    <td>Weak/Strong</td>
  </tr>
   <tr>
    <td>4</td>
    <td>BBa_J23106</td>
    <td>BBa_B0031</td>
    <td>Medium/Weak</td>
  </tr>
  <tr>
    <td>5</td>
    <td>BBa_J23106</td>
    <td>BBa_B0032</td>
    <td>Medium/Medium</td>
  </tr>
    <tr>
    <td>6</td>
    <td>BBa_J23106</td>
    <td>BBa_B0034</td>
    <td>Medium/Strong</td>
  </tr>
    <tr>
    <td>7</td>
    <td>BBa_J23119</td>
    <td>BBa_B0031</td>
    <td>Strong/Weak</td>
  </tr>
      <tr>
    <td>8</td>
    <td>BBa_J23119</td>
    <td>BBa_B0032</td>
    <td>Strong/Medium</td>
  </tr>
   <tr>
    <td>9</td>
    <td>BBa_J23119</td>
    <td>BBa_B0034</td>
    <td>Strong/Strong</td>
  </tr>
</table>
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<center> [[File:T--Lambert GA--tuningreference.png|800px]]

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<i>Figure 1: The highlighted points in yellow represent the combination of BBa_B0035 (reference RBS) with BBa_J23115 (reference promoter) compared to the remaining nine combinations highlighted in blue. Combinations showing promoters with the same strength, but different RBS, share similar expression. An increase in promoter strength results in an increase in expression; on the other hand, changes in RBS strength have a negligible effect on expression.</i> </center>
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<center>After we calculated Miller units, the data showed the reference’s level of expression was between the weak and medium promoter’s. As a result, the reference was graphed between the weak and medium promoter.</center>
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===Functional Parameters===
<partinfo>BBa_B0035 parameters</partinfo>
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