Difference between revisions of "Part:BBa J10054"
 
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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.  
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[[File:T--Lambert GA--tuningalldata.png|800px]]
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<style>
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table, th, td {
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  border: 1px solid black;
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  border-collapse: collapse;
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th, td {
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  padding: 15px;
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<i>Figure 1: The highlighted points in yellow represent BBa_J10050 (weak promoter/ weak RBS) 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 has a negligible effect on expression.</i>
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<table style="width:100%">
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<tr>
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    <th>Strain Identification Number </th>
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    <th>Promoter Part Number</th>
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    <th>RBS Part Number</th>
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    <th>Relative Strength of Promoter/RBS</th>
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  </tr>
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  <tr>
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    <td>R (positive control)</td>
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    <td>BBa_J23115</td>
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    <td>BBa_B0035</td>
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    <td>Reference/Reference</td>
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  </tr>
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  <tr>
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    <td>1</td>
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    <td>BBa_J23113</td>
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    <td>BBa_B0031</td>
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    <td>Weak/Weak</td>
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  </tr>
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  <tr>
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    <td>2</td>
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    <td>BBa_J23113</td>
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    <td>BBa_B0032</td>
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    <td>Weak/Medium</td>
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  </tr>
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  <tr>
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    <td>3</td>
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    <td>BBa_J23113</td>
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    <td>BBa_B0034</td>
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    <td>Weak/Strong</td>
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  </tr>
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  <tr>
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    <td>4</td>
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    <td>BBa_J23106</td>
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    <td>BBa_B0031</td>
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    <td>Medium/Weak</td>
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  </tr>
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  <tr>
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    <td>5</td>
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    <td>BBa_J23106</td>
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    <td>BBa_B0032</td>
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    <td>Medium/Medium</td>
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  </tr>
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    <tr>
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    <td>6</td>
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    <td>BBa_J23106</td>
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    <td>BBa_B0034</td>
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    <td>Medium/Strong</td>
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  </tr>
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    <tr>
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    <td>7</td>
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    <td>BBa_J23119</td>
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    <td>BBa_B0031</td>
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    <td>Strong/Weak</td>
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  </tr>
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      <tr>
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    <td>8</td>
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    <td>BBa_J23119</td>
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    <td>BBa_B0032</td>
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    <td>Strong/Medium</td>
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  </tr>
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  <tr>
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    <td>9</td>
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    <td>BBa_J23119</td>
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    <td>BBa_B0034</td>
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    <td>Strong/Strong</td>
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  </tr>
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</table>
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</body>
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</html>
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<br>
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<center>[[File:T--Lambert GA--tuning5highlighted.png|800px]]</center>
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<br>
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<center><i>Figure 1: The highlighted points in yellow represent BBa_J10054 (medium promoter/ medium RBS) 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>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 17:48, 21 October 2019


med promoter + med RBS + lacZa.GFP fusion

Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Medium constitutive promoter, medium 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 940

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
R (positive control) BBa_J23115 BBa_B0035 Reference/Reference
1 BBa_J23113 BBa_B0031 Weak/Weak
2 BBa_J23113 BBa_B0032 Weak/Medium
3 BBa_J23113 BBa_B0034 Weak/Strong
4 BBa_J23106 BBa_B0031 Medium/Weak
5 BBa_J23106 BBa_B0032 Medium/Medium
6 BBa_J23106 BBa_B0034 Medium/Strong
7 BBa_J23119 BBa_B0031 Strong/Weak
8 BBa_J23119 BBa_B0032 Strong/Medium
9 BBa_J23119 BBa_B0034 Strong/Strong


T--Lambert GA--tuning5highlighted.png


Figure 1: The highlighted points in yellow represent BBa_J10054 (medium promoter/ medium RBS) 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.