Team:SDU-Denmark/Tour41

From 2014.igem.org

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<h3>Expressions</h3>
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<h3>Expressions</h3><br>
<h4>Characerization of TetR/pTet</h4>
<h4>Characerization of TetR/pTet</h4>
<p>
<p>
-
<span class="intro">We wanted to test</span> if the Tet promoter could be fine-tuned, and what influence the LVA tag on TetR has on  
+
<span class="intro">As mentioned on the System design page</span>, we wanted to investigate the Tet promoter expression profile, and what influence the LVA tag on the pTet promoter regulator (TetR) had on the expression profile. <br><br>
-
the expression.<br><br>
+
-
The ligation of the TetR (+LVA) construct with the pTet-GFP construct was cloned and is found as
+
<span class="intro">For this purpose</span> three plasmids were constructed. The first expressing GFP from pTet promoter with no regulation. The second and third constitutively expressing TetR with or without the LVA tag as well<br><br>
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<a href="http://parts.igem.org/Part:BBa_K1475006" target="_blank">Bba_K1475006</a>.<br><br>
+
-
<span class="intro">In order for us</span> to ligate the TetR (no LVA) construct with the pTet-GFP construct, we first needed
+
<span class="intro">The pTet-GFP construct</span> was cloned.<br><br>
-
to remove the LVA tag from TetR. The ligation was cloned into a plasmid and can be
+
-
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475003" target="_blank">Bba_K1475003</a>.<br><br>
+
-
The ligation of TetR (no LVA) construct with the pTet-GFP construct was cloned and can
+
<span class="intro">A TetR (no LVA) construct</span> was cloned by PCR amplification without the LVA tag and addition of promoter, RBS and terminator. Subsequently the construct was ligated into the pTet-GFP construct. The constructs can be  
-
be found as <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>.<br><br>
+
found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475004" target="_blank">Bba_K1475004</a> and <a href="http://parts.igem.org/Part:Bba_K1475005" target="_blank">Bba_K1475005</a>, respectively.<br><br>
-
</p>
+
 
 +
<span class="intro">A TetR (with LVA) construct</span> was cloned by PCR amplification with the LVA tag and addition of the same promoter, RBS and terminator as TetR without LVA tag. Subsequently the construct was ligated into the pTet-GFP construct, as well. The construct can be found in parts registry as <a href="http://parts.igem.org/Part:Bba_K1475006" target="_blank">Bba_K1475006</a>.<br><br>
-
<h4>Characteriztion/expression</h4>
+
<h4>Characterization/expression</h4>
<p>
<p>
-
<span class="intro">The promoters in the</span> TetR-pTet constructs are supposed to be inhibited by TetR. By induction with
+
<span class="intro">The promoters in</span> the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed  
doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed  
after induction with  
after induction with  
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Therapy, 2007. 15:5, p. 938-945.
Therapy, 2007. 15:5, p. 938-945.
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br>
<a href="http://www.nature.com/mt/journal/v15/n5/full/6300118a.html" target="_blank"> (Link)</a></span><br><br>
-
To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS
+
<span class="intro">To test if the Tet promoter</span> could be fine-tuned using different concentrations of doxycycline, we ran FACS
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with  
(Fuorescence-activated Cell Sorting) on <i>E. coli</i> expressing GFP controlled by pTet, regulated by TetR with  
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and without LVA tag. A wild-type was used as control.
+
and without LVA tag. A wild-type was used as control.<br><br>
</p>
</p>
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<a class="popupImg alignCenter" style="width:500px" target="_blank"  href="http://2014.igem.org/wiki/images/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the FACS before and after induction with doxycycline.">
+
<a class="popupImg alignCenter" style="width:500px" target="_blank"  href="http://2014.igem.org/wiki/images/7/72/2014SDUexpressions1.png" title="Figure 1: Results of the fluorescence activated cell sorting (FACS) before and after induction with doxycycline. The strains used are NoTetR=E. coli K12 MG1655 with BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR:LVA=E. coli K12 MG1655 with BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter.">
   <img src="http://2014.igem.org/wiki/images/7/72/2014SDUexpressions1.png" style="width:500px" />
   <img src="http://2014.igem.org/wiki/images/7/72/2014SDUexpressions1.png" style="width:500px" />
Figure 1: Results of the FACS before and after induction with doxycycline.
Figure 1: Results of the FACS before and after induction with doxycycline.
</a>
</a>
<p>
<p>
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<span class="intro">The results of the</span> FACS illustrates that without induction with doxycycline, GFP is still expressed. This is
+
<br>
-
because the promoter is leaky. There is a very little variation in expression of GFP upon induction with low  
+
<span class="intro"> Comparing only the strains expressing either variants of TetR</span>, the results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. Most likely because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is markedly reduced in the constructs containing TetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA. <br><br>
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concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that  
+
-
TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA.<br><br>
+
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<span class="intro">Leaving the leakiness of</span> pTet out of account, FACS results indicate that the pTet inhibited by TetR with LVA  
+
<span class="intro">Although the FACS results indicates</span> that the pTet inhibited by TetR with LVA tag is the most responsive upon induction by doxycycline, we argue that the effect seen is due to overexpressing of TetR repressor. The hypothesis is based on the poor median fluorescence compared to un-regulated pTet promoter, even at doxycycline concentrations inhibitory of cell growth. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins. This could explain the better response from induction of TetR with LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without, supporting this hypothesis.<br><br>
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tag is the one most active, upon induction by doxycycline. This could be because the LVA tag makes TetR
+
-
more unstable and tags it for degredation or because TetR with LVA is better inhibited by doxycycline than TetR without LVA.<br><br>
+
-
To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was
+
<span class = "intro">By using a strain,</span> constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: <a href="http://parts.igem.org/Part:BBa_R0040:Experience" target="_blank">BBa_R0040:Experience</a> that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer, futher supporting the hypothesis.
 +
<br><br>
 +
 
 +
<span class = "intro">To analyse the amount</span> of TetR with and without LVA tag present in the cell, coomassie stainging was
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL,  
made on a SDS-page with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL,  
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR  
1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR  
(+LVA)-GFP, respectively.<br><br>
(+LVA)-GFP, respectively.<br><br>
-
<a class="popupImg alignCenter" style="width:500px" target="_blank"  href="http://2014.igem.org/wiki/images/2/23/2014SDUexpressions32.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.">
+
<a class="popupImg alignCenter" style="width:500px" target="_blank"  href="http://2014.igem.org/wiki/images/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" title="Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.">
-
   <img src="http://2014.igem.org/wiki/images/2/23/2014SDUexpressions32.png" style="width:500px" />
+
   <img src="http://2014.igem.org/wiki/images/b/be/SDU2014expressionsCoomassie_TetR_rettet_2000.png" style="width:500px" />
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.
Figure 2: Coomassie staining on with <i>E. coli</i> K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.
</a>
</a>
-
 
+
<br>
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the
<span class="intro">The coomassie staining shows</span> that the construct expressing TetR(+LVA) expresses more GFP than the
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA)  
construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA)  
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expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the  
expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the  
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due  
higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due  
-
to the LVA tag making TetR unstable and tagging it for degredation. <br><br>
+
to the LVA tag making TetR unstable and tagging it for degradation. <br><br>
-
Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to  
+
<span class = "intro">Because pTet is leaky,</span> all cells express GFP. It can be difficult to tell if the pTet has been induced and to  
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS  
what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS  
clearly shows an induction.<br><br>
clearly shows an induction.<br><br>
-
<span class="intro">Duplicates of plates with</span> doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500  
+
<span class="intro">Duplicates of plates with doxycycline</span> were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500  
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet  
ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet  
-
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated.
+
construct with no LVA, pTet-GFP without TetR construct and wild-type were plated. <br><br>
</p>
</p>
 +
<div>
<table frame="box" rules="rows" style="width:800px">
<table frame="box" rules="rows" style="width:800px">
   <tr>
   <tr>
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   </tr>
   </tr>
</table>
</table>
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<br><br>
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<span class = "intro">Table 1:</span> Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: <a href="http://parts.igem.org/Part:BBa_K1475005">BBa_K1475005</a> and <a href="http://parts.igem.org/Part:BBa_K1475006">BBa_K1475006</a>.
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<table frame="box" rules="rows" style="width:800px">
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</div>
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  <tr>
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<br>
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    <td style="width:200px"></td>
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    <td style="width:200px"> 0 ng/mL doxycycline</td>
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    <td style="width:200px"> 50 ng/mL doxycycline</td>
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    <td style="width:200px"> 100 ng/mL doxycycline</td>
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  </tr>
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  <tr>
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    <td style="width:200px">Second series of plating of <br>TetR-GFP at different concentrations of doxycycline<br><br><br><br><br><br><br></td>
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    <td style="width:200px">
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<a class="popupImg alignCenter" style="width:180px" target="_blank"  href="http://2014.igem.org/wiki/images/b/ba/2014SDUexpressions23.png" title="Plating of TetR-GFP at 0ng/mL doxycycline.">
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  <img src="http://2014.igem.org/wiki/images/4/41/2014SDUexpressions16.png" style="width:180px" />
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</a>
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    </td>
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    <td style="width:200px">
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<a class="popupImg alignCenter" style="width:180px" target="_blank"  href="http://2014.igem.org/wiki/images/4/48/2014SDUexpressions24.png" title="Plating of TetR-GFP at 50ng/mL doxycycline.">
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  <img src="http://2014.igem.org/wiki/images/7/70/2014SDUexpressions17.png" style="width:180px" />
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</a>
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    </td>
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    <td style="width:200px">
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<a class="popupImg alignCenter" style="width:180px" target="_blank"  href="http://2014.igem.org/wiki/images/5/5e/2014SDUexpressions25.png" title="Plating of TetR-GFP at 100ng/mL doxycycline.">
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  <img src="http://2014.igem.org/wiki/images/d/d3/2014SDUexpressions18.png" style="width:180px" />
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</a>
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    </td>
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  </tr>
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  <tr>
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    <td style="width:200px">200 ng/mL doxycycline</td>
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    <td style="width:200px">500 ng/mL doxycycline</td>
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    <td style="width:200px">1000 ng/mL doxycycline</td>
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    <td style="width:200px">2000 ng/mL doxycycline</td>
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  </tr>
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<tr>
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    <td style="width:200px">
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<a class="popupImg alignCenter" style="width:180px" target="_blank"  href="http://2014.igem.org/wiki/images/1/1f/2014SDUexpressions26.png" title="Plating of TetR-GFP at 200ng/mL doxycycline.">
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  <img src="http://2014.igem.org/wiki/images/4/40/2014SDUexpressions19.png" style="width:180px" />
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-
</a>
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    </td>
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    <td style="width:200px">
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<a class="popupImg alignCenter" style="width:180px" target="_blank"  href="http://2014.igem.org/wiki/images/1/1b/2014SDUexpressions27.png" title="Plating of TetR-GFP at 500ng/mL doxycycline.">
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  <img src="http://2014.igem.org/wiki/images/7/70/2014SDUexpressions20.png" style="width:180px" />
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</a>
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    </td>
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    <td style="width:200px">
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<a class="popupImg alignCenter" style="width:180px" target="_blank"  href="http://2014.igem.org/wiki/images/7/7b/2014SDUexpressions28.png" title="Plating of TetR-GFP at 1000ng/mL doxycycline.">
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  <img src="http://2014.igem.org/wiki/images/1/1e/2014SDUexpressions21.png" style="width:180px" />
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</a>
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    </td>
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    <td style="width:200px">
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<a class="popupImg alignCenter" style="width:180px" target="_blank"  href="http://2014.igem.org/wiki/images/b/b1/2014SDUexpressions29.png" title="Plating of TetR-GFP at 2000ng/mL doxycycline.">
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  <img src="http://2014.igem.org/wiki/images/7/76/2014SDUexpressions22.png" style="width:180px" />
+
-
</a>
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    </td>
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  </tr>
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</table><br>
+
<p>
<p>
-
<span class="intro">The plating of</span> TetR-GFP constructs on plates with doxycycline shows that GFP is expressed at different  
+
<span class="intro">The plating of TetR-GFP constructs</span> on plates with doxycycline shows that GFP is expressed at different  
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline  
levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline  
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This  
concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This  
-
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it could be seen in the FACS  
+
indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it was also seen from the FACS  
-
results.<br><br>
+
results. Furthermore, the plating assay proves that the bricks are functional, however slowly responding to induction (continuous induction over 24 hours compared to induction over 1 hour)<br><br>
-
<span class="intro">To see how the</span> growth of the bacteria expressing GFP controlled by pTet are affected, we have measured  
+
<span class="intro">To see how the growth</span> of the bacteria expressing GFP controlled by pTet are affected, we measured  
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-
OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)-
-
GFP, pTet (+LVA)-GFP and a wild-type.<br>
+
GFP, pTet (+LVA)-GFP and a wild-type.<br><br>
</p>
</p>
<div class="popupImg alignCenter" style="width:800px">
<div class="popupImg alignCenter" style="width:800px">
-
   <img src="http://2014.igem.org/wiki/images/e/e3/2014SDUexpressions31.png" style="width:800px" />
+
   <img src="http://2014.igem.org/wiki/images/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:800px" />
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.
Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.
</div>
</div>
<p>
<p>
-
<span class="intro">Figure 3 shows the</span> growth of bacteria expressing GFP constitutiely, are attenuated the most with most  
+
<br>
 +
<span class="intro">Figure 3 shows the growth</span> of bacteria expressing GFP constitutiely, are attenuated the most with most  
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the  
comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the  
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR
bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR  
with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR  
-
without LVA.</p><br><br>
+
without LVA.</p><br>
<h4>Characterization of lacI/plac</h4>
<h4>Characterization of lacI/plac</h4>
<p>
<p>
-
<span class="intro">2013 SDU-Denmark iGEM team</span> proved that the natural lac inhibitor has a faster respondance on induction  
+
<span class="intro">2013 SDU-Denmark iGEM team proved</span> that the natural lac inhibitor has a faster respondance on induction  
by IPTG, than lacI with LVA (<a href="http://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted  
by IPTG, than lacI with LVA (<a href="http://2013.igem.org/Team:SDU-Denmark/Tour52">Link</a>). As for pTet, we wanted  
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without  
to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without  
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully  
LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully  
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.
and can be found as <a href="http://parts.igem.org/Part:BBa_K1475007">Bba_K1475007</a>. Due to time constrains, we were never able to characterize this part and compare it to pTet.
-
 
+
<br><br><br>
</p>
</p>

Latest revision as of 03:57, 18 October 2014

Expressions


Characerization of TetR/pTet

As mentioned on the System design page, we wanted to investigate the Tet promoter expression profile, and what influence the LVA tag on the pTet promoter regulator (TetR) had on the expression profile.

For this purpose three plasmids were constructed. The first expressing GFP from pTet promoter with no regulation. The second and third constitutively expressing TetR with or without the LVA tag as well

The pTet-GFP construct was cloned.

A TetR (no LVA) construct was cloned by PCR amplification without the LVA tag and addition of promoter, RBS and terminator. Subsequently the construct was ligated into the pTet-GFP construct. The constructs can be found in parts registry as Bba_K1475004 and Bba_K1475005, respectively.

A TetR (with LVA) construct was cloned by PCR amplification with the LVA tag and addition of the same promoter, RBS and terminator as TetR without LVA tag. Subsequently the construct was ligated into the pTet-GFP construct, as well. The construct can be found in parts registry as Bba_K1475006.

Characterization/expression

The promoters in the TetR-pTet constructs are supposed to be inhibited by TetR. By induction with doxycycline, the repressor is inhibited, and thus pTet will be active. In this case, GFP will be expressed after induction with doxycycline. Source: Aagaard, L., et al.: A Facile Lentiviral Vector System for Ekspression of Doxycycline-Inducible dhRNAs: Knockdown of the Pre-miRNA Processing Enzyme Drosha. Molecular Therapy, 2007. 15:5, p. 938-945. (Link)

To test if the Tet promoter could be fine-tuned using different concentrations of doxycycline, we ran FACS (Fuorescence-activated Cell Sorting) on E. coli expressing GFP controlled by pTet, regulated by TetR with and without LVA tag. A wild-type was used as control.

Figure 1: Results of the FACS before and after induction with doxycycline.


Comparing only the strains expressing either variants of TetR, the results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. Most likely because the promoter is leaky. Despite 100% of the cells being fluorescent in the absence of doxycycline one can see that the fluorescence intensity is markedly reduced in the constructs containing TetR repressor. There is a very little variation in expression of GFP upon induction with low concentration of doxycycline. At high concentration of doxycycline (2000 ng/mL) it can clearly be seen that TetR (+LVA) inhibits pTet at a weaker extent than TetR without LVA.

Although the FACS results indicates that the pTet inhibited by TetR with LVA tag is the most responsive upon induction by doxycycline, we argue that the effect seen is due to overexpressing of TetR repressor. The hypothesis is based on the poor median fluorescence compared to un-regulated pTet promoter, even at doxycycline concentrations inhibitory of cell growth. pSB1C3 being a high copy plasmid leads to a high number of repressors, thus a higher concentration of doxycycline in needed to induce the expression from pTet. The LVA tag destabilizes TetR thus lovering the number of TetR proteins. This could explain the better response from induction of TetR with LVA. It can be seen from the coomassie stain below that there is less TetR repressor with LVA than without, supporting this hypothesis.

By using a strain, constitutively expressing tetR with pTet on a low copy plasmid UNIPV-Pavia iGEM 2011 shows here: BBa_R0040:Experience that pTet can be induced by aTc. Thus less TetR repressors in comparison to pTet sites increases the response to inducer, futher supporting the hypothesis.

To analyse the amount of TetR with and without LVA tag present in the cell, coomassie stainging was made on a SDS-page with E. coli K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.

Figure 2: Coomassie staining on with E. coli K12 (induced by 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline) expressing pTet-GFP, pTet-TetR (no LVA)-GFP and pTet-TetR (+LVA)-GFP, respectively.
The coomassie staining shows that the construct expressing TetR(+LVA) expresses more GFP than the construct expressing TetR(no LVA). In addition to this, the staining shows a higher amount of TetR(no LVA) in the cell than of TetR(+LVA). This is consistent with the FACS results that illustrates that pTet-TetR(+LVA) expresses more GFP than pTet-TetR(no LVA). The coomassie staining indicates that the reason for the higher expression of GFP by pTet-TetR (+ LVA) is because the cell contains less inhibitor. This must be due to the LVA tag making TetR unstable and tagging it for degradation.

Because pTet is leaky, all cells express GFP. It can be difficult to tell if the pTet has been induced and to what extent, however, plates containing the corresponding concentrations of doxycycline as used in FACS clearly shows an induction.

Duplicates of plates with doxycycline were made with 0 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL and 2000 ng/mL doxycycline. On the plates, TetR-pTet construct with LVA, TetR-pTet construct with no LVA, pTet-GFP without TetR construct and wild-type were plated.

0 ng/mL doxycycline 50 ng/mL doxycycline 100 ng/mL doxycycline
First series of plating of
TetR-GFP at different concentrations of doxycycline






200 ng/mL doxycycline 500 ng/mL doxycycline 1000 ng/mL doxycycline 2000 ng/mL doxycycline
Table 1: Plating of E. coli MG1655 K12 expressing different constructs on plates containing a varying concentration of doxycycline: GFP=BBa_K136030, GFP regulated by the constitutively active p(tetR). tetR no LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor without the LVA-tag and the p(tetR) promoter. tetR +LVA=BBa_K1475005, GFP controlled by a constitutively expressed tetR repressor with the LVA-tag and the p(tetR) promoter. The experiment was done in duplicates but the second line of results was omitted from this wiki because the results showed the same, please see the parts registry page for all results: BBa_K1475005 and BBa_K1475006.

The plating of TetR-GFP constructs on plates with doxycycline shows that GFP is expressed at different levels at different concentrations of doxycycline. Expression increases with an increase in doxycycline concentrations. The plates also show that GFP, to some extent, is expressed without doxycycline. This indicates that the Tet promoter is leaky and is not fully inhibited by TetR as it was also seen from the FACS results. Furthermore, the plating assay proves that the bricks are functional, however slowly responding to induction (continuous induction over 24 hours compared to induction over 1 hour)

To see how the growth of the bacteria expressing GFP controlled by pTet are affected, we measured OD over 8 hours. We measured OD on triplicates of bacteria with an empty vector, pTet-GFP, pTet (no LVA)- GFP, pTet (+LVA)-GFP and a wild-type.

Figure 3: Growth curve of bacteria expressing pTet (+LVA)-GFP, pTet (no LVA)-GFP, pTet-GFP, an empty vector and a wild-type.


Figure 3 shows the growth of bacteria expressing GFP constitutiely, are attenuated the most with most comprised growth. Removing the LVA tag from TetR also has a negative effect on the growth of the bacteria. This could be because TetR without LVA stresses the metabolism of the bacteria more than TetR with LVA or because LVA tags TetR for degradation and thus TetR with LVA stresses the cell less than TetR without LVA.


Characterization of lacI/plac

2013 SDU-Denmark iGEM team proved that the natural lac inhibitor has a faster respondance on induction by IPTG, than lacI with LVA (Link). As for pTet, we wanted to test if the lac promoter could be fine-tuned. Due to the 2013 SDU iGEM team, we used the lacI without LVA. We wanted to ligate a constitutive promoter-lacI (no LVA) with plac-GFP. This was done successfully and can be found as Bba_K1475007. Due to time constrains, we were never able to characterize this part and compare it to pTet.