Difference between revisions of "Part:BBa B0035"
 
(5 intermediate revisions by 3 users not shown)
Line 13: Line 13:
 
==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.
−
 
 
<br>
 
<br>
 +
<br>
 +
<html>
 +
 +
<style>
 +
table, th, td {
 +
  border: 1px solid black;
 +
  border-collapse: collapse;
 +
}
 +
th, td {
 +
  padding: 15px;
 +
}
 +
</style>
 +
</head>
 +
<body>
 +
 +
<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>
 +
</body>
 +
</html>
 +
  
 
<center> [[File:T--Lambert GA--tuningreference.png|800px]]
 
<center> [[File:T--Lambert GA--tuningreference.png|800px]]
  
−
Figure 1: The highlighted points in yellow represent BBa_B0035 (reference 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. </center>
+
<br>
  
 +
<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>
 +
<br>
 +
<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>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_B0035 parameters</partinfo>
 
<partinfo>BBa_B0035 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 20:19, 21 October 2019


RBS (B0030 derivative)

-- No description --

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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--tuningreference.png


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.


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.