View source for Part:BBa B0034:Experience

__NOTOC__
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
how you used this part and how it worked out.

===Characterizations of BBa_B0034===
The Alverno_Ca team characterized BBa_K2066035, which contains BBa-B0034, as well as a series of other plasmids using TX-TL, a mean of in vitro transcription and translation using cell extract. Below is a graph of normalized expression of BBa_K2066035 in comparison to the other palsmids in the sequence. 
https://static.igem.org/mediawiki/2016/4/4f/T--Alverno_CA--W%26M_corrected.png

This graph shows the fluorescence of the plasmids, over a period of 12 and a half hours, in a plate reader.
https://static.igem.org/mediawiki/2016/c/c1/T--Alverno_CA--W%26M_plasmid_time_traces_in_TXTL%282%29.png"

===Applications of BBa_B0034===

===User Reviews===
<!-- DON'T DELETE --><partinfo>BBa_B0034 StartReviews</partinfo>
<!-- Template for a user review
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview number</partinfo>
<I>Username</I>
|width='60%' valign='top'|
Enter the review inofrmation here.
|};
<!-- End of the user review template -->

{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>iGEM Team Göttingen 2013</I>
|width='60%' valign='top'|
We used the part for our reporter system and it worked very good! We also improved it by switching the pre- and suffix, basically inverting it. This way, we were able to use it in an "inverted" expression unit on the same vector as our reporter system. For further information see: https://parts.igem.org/wiki/index.php?title=Part:BBa_K1045010
|};
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>2012 iGEM UIUC</I>
|width='60%' valign='top'|
The part worked as expected when used with BBa_E0030. Read additional results at http://2012.igem.org/Team:UIUC-Illinois/Results.
|}
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>Antiquity</I>
|width='60%' valign='top'|
This review comes from the old result system and indicates that this part worked in some test.
|}
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_R0051 AddReview 5</partinfo>
<I> Aberdeen_Scotland 2009 </I>
|width='60%' valign='top'|
Our miniprep, double digest and gel worked properly. We used this part in fusion PCR for building our biobrick <partinfo>BBa_K182102</partinfo>. The fusion PCR, cloning and sequence were all correct.
|}
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I> iGEM Groningen 2009 </I>
|width='60%' valign='top'|
The ligation of part <partinfo>BBa_K190028</partinfo> behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several genes for building our biobricks e.g. <partinfo>BBa_K190061</partinfo>.
|}

{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>maven</I>
|width='60%' valign='top'|
Defining efficiency for an RBS seems misleading, since RBS efficiency can only be considered in context of its upstream and downstream sequences as this [http://www.nature.com/nbt/journal/v27/n10/abs/nbt.1568.html article] demonstrated.
|}

{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>iGEM Copenhagen</I>
|width='60%' valign='top'|
We attempted to use this BioBrick to assembly a protein expressing device. We were unable to do it with standard assembly but had succesful result utilizing the 2011 [http://2011.igem.org/Team:Copenhagen/Collaboration| DTU-2 user assembly plug'n'play method]
|}

{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>iGEM Amsterdam 2011</I> 
|width='60%' valign='top'|
Our results show proteins can be expressed at low temperatures using this RBS. From this we conclude that the RBS is useful for translation in cold environments. 
|};
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>iGEM Team Nanjing-China 2024</I>
|width='60%' valign='top'|
We used the part for our reporter system and it worked very good!It can be used as a good RBS in Shewanella, which we have used extensively in our projects.
|};
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_E0040 AddReview 5</partinfo>
<I>iGEM Dundee 2013</I>
|width='60%' valign='top'|

The function of this part was characterised in a new salt-sensing device. This ribosome binding site (<partinfo>BBa_B0034</partinfo>) was linked up to GFP (<partinfo>BBa_E0040</partinfo>) under the control of an OmpR-responsive promoter (<partinfo>BBa_R0083</partinfo>). The new composite part (a reporter) (<partinfo>BBa_K1012005</partinfo>) was found to work as expected, inducing GFP production under osmotic stress conditions.
|}
;
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>iGEM Nanjing China 2024</I> 
|width='60%' valign='top'|
We developed RBS of different intensities in tandem with PPK1 and expressed them in <i>Shewanella</i>.This RBS element works well in Shewanella
<html>
<body>
    <div style="text-align: center;">
        <img src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-ppk1.png" style="width: 500px; height: auto;">
    </div>
</body>
</html>
Figure 2: Basic construction of PPK1 plasmid

The best results can be seen after adopting the RBS BBa-B0034, which is a function-based verification.
<html>
<body>
    <div style="text-align: center;">
        <img src="https://static.igem.wiki/teams/5034/engineering/fig11.png" style="width: 500px; height: auto;">
    </div>
</body>
</html>
Figure 3: Phosphorus accumulation capacity in <i>S. oneidensis.</i> after the introduction of PPK1
We also performed polyacrylamide gel electrophoresis and found that there was indeed the largest amount of PPK1 expression in tandem with the B0034 element, which was a direct validation based on protein expression.
<html>
<body>
    <div style="text-align: center;">
        <img src="https://static.igem.wiki/teams/5034/results/figure9.png" style="width: 500px; height: auto;">
    </div>
</body>
</html>
|};

<!-- DON'T DELETE --><partinfo>BBa_B0034 EndReviews</partinfo>





<!-- DON'T DELETE --><partinfo>BBa_B0034 StartReviews</partinfo>
<!-- Template for a user review
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview NUMBER</partinfo>
<I>Username</I>
|width='60%' valign='top'|
Enter the review inofrmation here.
|};
<!-- End of the user review template -->
{|width='80%' style='border:1px solid gray'
|-
|width='10%'|
<partinfo>BBa_B0034 AddReview 5</partinfo>
<I>UNIPV-Pavia iGEM 2011</I>
|width='60%' valign='top'|
<html>


<em>
NB: unless differently specified, all tests were performed in <a href='http://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="https://parts.igem.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>. 
</em>

<br><br>

<p align='justify'>
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. 
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p>
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p>
<ol>
<li>select the RBSs you want to study</li>
<li>assemble them in a Promoter - XX - Coding sequence circuit</li>

<table align='center' width='100%'>
<tr>
<td>
<div style='text-align:center'><div class="thumbinner" style="width:100%;">
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage"  width="60%"></a></div></div>
</td>
</tr>
</table>


<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='https://parts.igem.org/wiki/index.php/Part:	BBa_B0034'>BBa_B0034</a>). </li></div></ol><br>


<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p>
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built  to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p>
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p>
<br>
<div align="center"><table class='data' width='70%' border="1"><tr>
<td class='row'><b>RBS</b></td>
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td>
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td>
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td>
<td class='row'><b>Declared efficiency</b></td>
</tr><tr>
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td>
</tr><tr>
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td>
</tr><tr>
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td>
</tr><tr>
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td>
</tr></table></div>

<br>
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br>

<div align="center"><table class='data' width='70%' border="1"><tr>
<td class='row'><b>RBS</b></td>
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td>
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td>
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td>
<td class='row'><b>Declared efficiency</b></td>
</tr><tr>
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.96</td><td class='row'>0.45</td><td class='row'>0,6</td>
</tr><tr>
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.78</td><td class='row'>0.028</td><td class='row'>0,07</td>
</tr><tr>
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.51</td><td class='row'>N.D.</td><td class='row'>0.3</td>
</tr><tr>
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td>
</tr></table></div>
<br>
<p align='justify'>
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p>
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p>
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio between the percentage of degraded HSL after 7 hours of the studied system and the reference B0034 in <em>E. coli</em> TOP010, in high copy number plasmid pSB1A2.</p>
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='http://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div>

<br>
</p>
The parts we used to characterize the RBSs are listed here:
<ol>
<ul><li>mRFP expression with different promoters</li>
<ul>
<li>J23101</li>
<ul class="disc">
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516130	">	BBa_K516130	</a>	J101-RBS30-RFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516131	">	BBa_K516131	</a>	J101-RBS31-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516132	">	BBa_K516132	</a>	J101-RBS32-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_J23101	">	BBa_J23101	</a>	J101-RBS34-mRFP-TT	</li>
</ul>
<li>p<sub>Tet</sub></li>
<ul class="disc">
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516230	">	BBa_K516230	</a>	p<sub>Tet</sub>-RBS30-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516231	">	BBa_K516231	</a>	p<sub>Tet</sub>-RBS31-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516232	">	BBa_K516232	</a>	p<sub>Tet</sub>-RBS32-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_I13521	">	BBa_I13521	</a>	p<sub>Tet</sub>-RBS34-mRFP-TT	</li>
</ul>
<li>p<sub>Lux</sub></li>
<ul class="disc">
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516330	">	BBa_K516330	</a>	pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516331	">	BBa_K516331	</a>	pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516332	">	BBa_K516332	</a>	pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516334	">	BBa_K516334	</a>	pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT	</li>
</ul>
</ul>
<li>p<sub>Tet</sub> driving the expression of different genes</li>
<ul><li>mRFP</li>
<ul class="disc">
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516230	">	BBa_K516230	</a>	p<sub>Tet</sub>-RBS30-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516231	">	BBa_K516231	</a>	p<sub>Tet</sub>-RBS31-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516232	">	BBa_K516232	</a>	p<sub>Tet</sub>-RBS32-mRFP-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_I13521	">	BBa_I13521	</a>	p<sub>Tet</sub>-RBS34-mRFP-TT	</li>
</ul>
<li>AiiA</li>
<ul class="disc">
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516220	">	BBa_K516220	</a>	p<sub>Tet</sub>-RBS30-AiiA-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516221	">	BBa_K516221	</a>	p<sub>Tet</sub>-RBS31-AiiA-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516222	">	BBa_K516222	</a>	p<sub>Tet</sub>-RBS32-AiiA-TT	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516224	">	BBa_K516224	</a>	p<sub>Tet</sub>-RBS34-AiiA-TT	</li>
</ul>
<li>LuxI</li>
<ul class="disc">
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516210	">	BBa_K516210	</a>	p<sub>Tet</sub>-RBS30-LuxI	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516211	">	BBa_K516211	</a>	p<sub>Tet</sub>-RBS31-LuxI	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516212	">	BBa_K516212	</a>	p<sub>Tet</sub>-RBS32-LuxI	</li>
<li>	<A HREF="https://parts.igem.org/wiki/index.php/Part:	BBa_K516214	">	BBa_K516214	</a>	p<sub>Tet</sub>-RBS34-LuxI	</li>
</ul></ul>
</ul></ol>


</p>
<p align='justify'>
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br>
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while  with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. 
</p>
<p align='justify'>
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:
<ul>
<li>low efficiency RBS (B0031)</li>
<li>medium efficiency RBS (B0032)</li>
<li>high efficiency RBSs (B0030, B0034)</li>
</ul> 
</p>


</html>
|}

<!-- DON'T DELETE --><partinfo>BBa_B0034 EndReviews</partinfo>