Difference between revisions of "Team:Hong Kong HKUST/Further"

 
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     <h1 class="title text-center">Altering Gene expression by Changing Copy Number of Vector Plasmid<br><p style="font-size:35px;"></p></h1>
 
     <h1 class="title text-center">Altering Gene expression by Changing Copy Number of Vector Plasmid<br><p style="font-size:35px;"></p></h1>
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   <blockquote><p>One of the major challenge of the Tristable switch is balancing gene expression levels. To achieve this goal, we target the copy number of expression vector. We exploit the natural mechanism that control R6K plasmid replication. <br>
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   <blockquote><p>A major challenge of the Tristable Switch is balancing the expression level of each gene. To achieve this goal, we targeted the copy number of expression vector through the exploitation of a natural mechanism, in an attempt to control R6K plasmid replication.<br>
  
 
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<td colspan="2"><p>Pi-protein controls the initiation of DNA replication in gamma origin of R6K plasmid. By varying the amount of Pi-protein being produced through an inducible promoter (ptet*), the change in plasmid copy number can be observed using Real Time PCR.
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<td colspan="2"><p>Pi-protein controls the initiation of DNA replication in gamma origin of R6K plasmid. By varying the amount of Pi-protein being produced through an inducible promoter (ptet*), the change in plasmid copy number can be observed using real-time PCR.
 
This method is being studied because it could potentially offer a linear change of gene expression. <br>
 
This method is being studied because it could potentially offer a linear change of gene expression. <br>
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Also this method provides alternative means to control gene expression, other than controlling gene expression through the use of promoters and ribosomal binding sites (RBS), thus expanding the degree of control over gene expression. </p></td>
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Also, this method provides an alternative means to control gene expression, other than controlling gene expression through the use of promoters and ribosomal binding sites (RBS); thus expanding the degree of control over gene expression. </p></td>
 
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   <h2 class="text-muted"><em><b>Results</b></em></h2><br>
 
   <h2 class="text-muted"><em><b>Results</b></em></h2><br>
 
<img class="img-responsive" src= "https://static.igem.org/mediawiki/2016/a/ac/T--Hong_Kong_HKUST--R6K_real_time.png" style="width:85%; margin-left:auto; margin-right:auto;"><br><br>
 
<img class="img-responsive" src= "https://static.igem.org/mediawiki/2016/a/ac/T--Hong_Kong_HKUST--R6K_real_time.png" style="width:85%; margin-left:auto; margin-right:auto;"><br><br>
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<Figp style="font-size:14px; padding: 0 0.9em;"><b>Characterization of plasmid controlling system.</b> (A) Pir gene and gamma ORI co-exist on the same plasmid. (B) Pir gene and gamma ORI exist on different plasmids. Error bars represent STD.
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<Figp style="font-size:14px; padding: 0 0.9em;"><b>Characterization of plasmid controlling system.</b> (A) Pir gene and gamma ORI co-exist on the same plasmid. (B) Pir gene and gamma ORI exist on different plasmids.  
 
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<p>From the above figure, no significant change in copy number of plasmid (per genome) was observed upon induction by aTc when the gamma ORI and pir gene are located on different plasmids. <br><br>
 
<p>From the above figure, no significant change in copy number of plasmid (per genome) was observed upon induction by aTc when the gamma ORI and pir gene are located on different plasmids. <br><br>
  
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We speculate that this might due to the high sensitivity of R6K ORI to pi protein. As reported by previous study, the overexpression of pir gene facilitates the formation of dimeric pi protein that conversely suppress the copy number of plasmid with gamma origin. (Abhyankar, 2003) this can be simply understood as neither too low nor too high concentration of pi protein will increase the plasmid copy number. It is conjectured that the range of [aTc] that would actually affect the copy number is very narrow and it is, therefore, hidden in one of the 10-fold interval of the [aTc]. Our experimental setups might have only shown the lowest copy number of p(R6K)-BBa_J04450 possible.
+
We speculate that this might be due to the high sensitivity of R6K ORI to pi protein. As reported by previous study, the overexpression of pir gene facilitates the formation of dimeric pi protein that conversely suppress the copy number of plasmid with gamma origin (Abhyankar, 2003). This can be simply understood as neither too high nor too low the concentration of pi protein will the plasmid copy number be increased. It is conjectured that there only exists a very narrow range of [aTc], which would affect the copy number; and that the range is hidden in one of the 10-fold interval of [aTc], regardless, our experimental setups might have shown the lowest possible copy number of p(R6K)-BBa_J04450.
 
<br><br>Abhyankar, M. M. (2003). Biochemical Investigations of Control of Replication Initiation of Plasmid R6K. <i>Journal of Biological Chemistry</i>, <i>279</i>(8), 6711–6719.  
 
<br><br>Abhyankar, M. M. (2003). Biochemical Investigations of Control of Replication Initiation of Plasmid R6K. <i>Journal of Biological Chemistry</i>, <i>279</i>(8), 6711–6719.  
 
</p><br><br>
 
</p><br><br>
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   <p>As for p(R6K)-TPP-BBa_J04450, the result showed that there is a significant increase in plasmid copy number when the cells are induced with 10<sup>-3</sup> μM aTc. however, a step-wise increment was not observed. Therefore, more measurement point on that particular range of [aTc], which is from 10<sup>-2</sup> μM to 10<sup>-4</sup> μM aTc, have to be set up in order to confirm if the plasmid copy number will raise progressive as expected.</p>
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   <p>As for p(R6K)-TPP-BBa_J04450, the result showed that there was a significant increase in plasmid copy number when the cells were induced with 10<sup>-3</sup> μM aTc. However, a step-wise increment was not observed. Therefore, more measurement point on that particular range of [aTc], which is from 10<sup>-2</sup> μM to 10<sup>-4</sup> μM aTc, has to be set up in order to confirm whether or not the plasmid copy number raise progressively.</p>
 
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<h2 class="text-muted"><em><b>Conclusion</b></em></h2><br>
 
<h2 class="text-muted"><em><b>Conclusion</b></em></h2><br>
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<p>The plasmid copy number of p(R6K)-TPP-BBa_J04450 remains constant across different [aTc] while that of p(R6K)-BBa_J04450 was only elevated when induced with 10<sup>3</sup> uM [aTc],in our experimental context. Pinpointing the correct range of [aTc] that affects the plasmid copy number as well as removing the additional sequence would serve as further investigation to confirm the cause for the unvarying plasmid copy number.
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<p>The plasmid copy number of p(R6K)-TPP-BBa_J04450 remains constant across different [aTc] while that of p(R6K)-BBa_J04450 was only elevated when induced with 10<sup>3</sup> μM [aTc],in our experimental context. Pinpointing the correct range of [aTc] that affects the plasmid copy number would serve as further investigation to confirm the cause for the invariant plasmid copy number.
 
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  </p>  
 
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Latest revision as of 03:29, 20 October 2016

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Altering Gene expression by Changing Copy Number of Vector Plasmid

A major challenge of the Tristable Switch is balancing the expression level of each gene. To achieve this goal, we targeted the copy number of expression vector through the exploitation of a natural mechanism, in an attempt to control R6K plasmid replication.



Pi-protein controls the initiation of DNA replication in gamma origin of R6K plasmid. By varying the amount of Pi-protein being produced through an inducible promoter (ptet*), the change in plasmid copy number can be observed using real-time PCR. This method is being studied because it could potentially offer a linear change of gene expression.
Also, this method provides an alternative means to control gene expression, other than controlling gene expression through the use of promoters and ribosomal binding sites (RBS); thus expanding the degree of control over gene expression.


Results




Characterization of plasmid controlling system. (A) Pir gene and gamma ORI co-exist on the same plasmid. (B) Pir gene and gamma ORI exist on different plasmids.

From the above figure, no significant change in copy number of plasmid (per genome) was observed upon induction by aTc when the gamma ORI and pir gene are located on different plasmids.

We speculate that this might be due to the high sensitivity of R6K ORI to pi protein. As reported by previous study, the overexpression of pir gene facilitates the formation of dimeric pi protein that conversely suppress the copy number of plasmid with gamma origin (Abhyankar, 2003). This can be simply understood as neither too high nor too low the concentration of pi protein will the plasmid copy number be increased. It is conjectured that there only exists a very narrow range of [aTc], which would affect the copy number; and that the range is hidden in one of the 10-fold interval of [aTc], regardless, our experimental setups might have shown the lowest possible copy number of p(R6K)-BBa_J04450.

Abhyankar, M. M. (2003). Biochemical Investigations of Control of Replication Initiation of Plasmid R6K. Journal of Biological Chemistry, 279(8), 6711–6719.



As for p(R6K)-TPP-BBa_J04450, the result showed that there was a significant increase in plasmid copy number when the cells were induced with 10-3 μM aTc. However, a step-wise increment was not observed. Therefore, more measurement point on that particular range of [aTc], which is from 10-2 μM to 10-4 μM aTc, has to be set up in order to confirm whether or not the plasmid copy number raise progressively.

Conclusion


The plasmid copy number of p(R6K)-TPP-BBa_J04450 remains constant across different [aTc] while that of p(R6K)-BBa_J04450 was only elevated when induced with 103 μM [aTc],in our experimental context. Pinpointing the correct range of [aTc] that affects the plasmid copy number would serve as further investigation to confirm the cause for the invariant plasmid copy number.