Difference between revisions of "Part:BBa K4307006"

 
m
 
Line 3: Line 3:
 
<partinfo>BBa_K4307006 short</partinfo>
 
<partinfo>BBa_K4307006 short</partinfo>
  
−
Serine integrase, adapted from bacteriophage, is capable of catalyzing site-specific recombination between two attachment sites (attP and attB) and inverting the DNA sequence flanked by these two opposing sites. We use Bxb1 integrase, an integrase found in Mycobacteriophage Bxb1 in our project.  
+
Serine integrase, adapted from bacteriophage, is capable of catalyzing site-specific recombination between two attachment sites (attP and attB) and inverting the DNA sequence flanked by these two opposing sites. We use Bxb1 integrase, an integrase found in Mycobacteriophage Bxb1 in our project.  <br>
 +
 
 +
This time, we combined attP and attB, the attachment sites of serine integrase with OR1-OR2, the part that has promoter function in the right operator (OR) of phage genome.  
  
−
This time, we combined attP and attB, the attachment sites of serine integrase with OR1-OR2, the part that has promoter function in the right operator (OR) of phage genome.
 
  
  
Line 14: Line 15:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4307006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4307006 SequenceAndFeatures</partinfo>
 +
 +
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_K4307000 parameters</partinfo>
 +
<!-- -->
 +
 +
 +
 +
 +
<h2>Characterization</h2>
 +
<p>The following figure demonstrates our successful construction.</p>
 +
 +
<br>
 +
https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307006-gel.png<br>
 +
                <b>Figure 1: </b> <b>The construction results of attB-promoter-attP and Ser integrase.</b>
 +
           
 +
 +
<h3>Fluorescence assay was done to characterize the biobrick.</h3>
 +
<p>To ensure the inversion effect of Bxb1 integrase, we locate mCherry and EGFP upstream and downstream of OR1-OR2 promoter respectively.</p>
 +
<p>The gene of Bxb1 integrase was integrated with pBAD promoter. The fluorescence signal of mCherry was recorded at 16th hour after induction with 1% (w/v) arabinose in this experiment. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell. All groups were carried out twice to do a statistical analysis. Different experiments were induced in one 96 well plate. The culture temperature was 37℃ in this trial. </p>
 +
 +
<br>
 +
https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307006-1.png<br>
 +
                <b>Figure 2: </b> <b> Characterization of serine integrase function by fluorescence spectrophotometry.</b>
 +
         
 +
 +
 +
 +
 +
<h3>qPCR assay was done to characterize the biobrick. </h3>
 +
<p>We applied quantitative RT-PCR to measure the inversion efficiency of serine integrase. The primers used in qPCR were designed to amplify sequences in between only after inversion caused by integrase. We analyzed the inversion efficiency of serine integrase by measuring the Ct values of qPCR before and after arabinose induction. The influence of bacterial amount on the Ct values of target gene was blanked by measuring the Ct value of a reference gene (resistance marker gene on the plasmid vector).</p>
 +
 +
<p>After intricate analysis, we summarized valid data and performed regression analysis to test our detection. (Table 1) Even though the exact inversion efficiency cannot be determined, relative magnitude demonstrated that our system induced a two-fold increase in the ratio of inversed promoter sequence (Figure 3).</p>
 +
<br>
 +
 +
  <table>
 +
  <caption>Table 1: qPCR results of serine integrase characterization.</caption>
 +
 
 +
  <thead>
 +
        <th>Induction temperature (℃)</th>
 +
        <th>Induction time (h)</th>
 +
        <th>Target sequence Ct (unit)</th>
 +
        <th>Reference sequence Ct (unit) </th>
 +
       
 +
    </thead>
 +
   
 +
    <tr>
 +
      <td rowspan="2">Group 1</td>
 +
        <td>Induced</td>
 +
        <td>32.9</td>
 +
        <td>32.18 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Not induced</td>
 +
        <td>35.07</td>
 +
        <td>31.72 </td>
 +
    </tr>
 +
    <tr>
 +
      <td rowspan="2">Group 2</td>
 +
        <td>Induced</td>
 +
        <td>14.195</td>
 +
        <td>14.655 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Not induced</td>
 +
        <td>14.91</td>
 +
        <td>13.615 </td>
 +
    </tr>
 +
    <tr>
 +
      <td rowspan="2">Group 3</td>
 +
        <td>Induced</td>
 +
        <td>14.905</td>
 +
        <td>15.28 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Not induced</td>
 +
        <td>14.975</td>
 +
        <td>15.025 </td>
 +
    </tr>
 +
    <tr>
 +
      <td rowspan="2">Group 4</td>
 +
        <td>Induced</td>
 +
        <td>13.834</td>
 +
        <td>14.705 </td>
 +
    </tr>
 +
    <tr>
 +
        <td>Not induced</td>
 +
        <td>14.785</td>
 +
        <td>13.325 </td>
 +
    </tr>
 +
    <tr>
 +
      <td rowspan="2">Regression analysis</td>
 +
        <td>Induced</td>
 +
        <td colspan="2"> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;- 0.8791 * p + q - 0.8961 = 0</td>
 +
       
 +
    </tr>
 +
    <tr>
 +
        <td>Not induced</td>
 +
        <td colspan="2"> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;- 0.9299 * p + q - 1.5752 = 0</td>
 +
       
 +
    </tr>
 +
</table>
 +
 +
<br>
 +
https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307006-2.png<br>
 +
                <b>Figure 3: </b> <b>Characterization of serine integrase inversion efficiency by RT-qPCR. An exemplary qP graph (left) and the regression results (right).</b>
 +
               
 +
         
 +
 +
<h2>Conclusion</h2>
 +
<p>We successfully expressed the integrase from bacteriophage in bacteria, and the results of the fluorescence assay showed that integrase could have obvious inversion effect, but the action time was a little bit long.</p>
  
  

Latest revision as of 15:51, 12 October 2022


attB-promoter-attP & Ser integrase

Serine integrase, adapted from bacteriophage, is capable of catalyzing site-specific recombination between two attachment sites (attP and attB) and inverting the DNA sequence flanked by these two opposing sites. We use Bxb1 integrase, an integrase found in Mycobacteriophage Bxb1 in our project.

This time, we combined attP and attB, the attachment sites of serine integrase with OR1-OR2, the part that has promoter function in the right operator (OR) of phage genome.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 361
    Illegal PstI site found at 667
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 361
    Illegal NheI site found at 350
    Illegal PstI site found at 667
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 361
    Illegal BglII site found at 525
    Illegal BamHI site found at 289
    Illegal BamHI site found at 549
    Illegal BamHI site found at 927
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 361
    Illegal PstI site found at 667
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 361
    Illegal PstI site found at 667
    Illegal NgoMIV site found at 4
    Illegal NgoMIV site found at 1266
    Illegal AgeI site found at 124
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 24
    Illegal BsaI.rc site found at 1933
    Illegal SapI site found at 106




Characterization

The following figure demonstrates our successful construction.


bba-k4307006-gel.png

               Figure 1:  The construction results of attB-promoter-attP and Ser integrase.

Fluorescence assay was done to characterize the biobrick.

To ensure the inversion effect of Bxb1 integrase, we locate mCherry and EGFP upstream and downstream of OR1-OR2 promoter respectively.

The gene of Bxb1 integrase was integrated with pBAD promoter. The fluorescence signal of mCherry was recorded at 16th hour after induction with 1% (w/v) arabinose in this experiment. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell. All groups were carried out twice to do a statistical analysis. Different experiments were induced in one 96 well plate. The culture temperature was 37℃ in this trial.


bba-k4307006-1.png

               Figure 2:   Characterization of serine integrase function by fluorescence spectrophotometry.



qPCR assay was done to characterize the biobrick.

We applied quantitative RT-PCR to measure the inversion efficiency of serine integrase. The primers used in qPCR were designed to amplify sequences in between only after inversion caused by integrase. We analyzed the inversion efficiency of serine integrase by measuring the Ct values of qPCR before and after arabinose induction. The influence of bacterial amount on the Ct values of target gene was blanked by measuring the Ct value of a reference gene (resistance marker gene on the plasmid vector).

After intricate analysis, we summarized valid data and performed regression analysis to test our detection. (Table 1) Even though the exact inversion efficiency cannot be determined, relative magnitude demonstrated that our system induced a two-fold increase in the ratio of inversed promoter sequence (Figure 3).


<thead> 
   </thead>
Table 1: qPCR results of serine integrase characterization.
Induction temperature (℃) Induction time (h) Target sequence Ct (unit) Reference sequence Ct (unit)
Group 1 Induced 32.9 32.18
Not induced 35.07 31.72
Group 2 Induced 14.195 14.655
Not induced 14.91 13.615
Group 3 Induced 14.905 15.28
Not induced 14.975 15.025
Group 4 Induced 13.834 14.705
Not induced 14.785 13.325
Regression analysis Induced                     - 0.8791 * p + q - 0.8961 = 0
Not induced                     - 0.9299 * p + q - 1.5752 = 0


bba-k4307006-2.png

               Figure 3:  Characterization of serine integrase inversion efficiency by RT-qPCR. An exemplary qP graph (left) and the regression results (right).

Conclusion

We successfully expressed the integrase from bacteriophage in bacteria, and the results of the fluorescence assay showed that integrase could have obvious inversion effect, but the action time was a little bit long.