Difference between revisions of "Part:BBa C0061:Experience"

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<I>Yourid</I>
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<I>Youri</I>
 
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This part was used as a subpart in [https://parts.igem.org/Part:BBa_K546005 K546005] and found to be functional.
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This part was used as a subpart in [https://parts.igem.org/Part:BBa_K546005 K546005], [https://parts.igem.org/Part:BBa_K546546 K546546] and found to be functional.
  
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https://parts.igem.org/Part:BBa_K2267027We improved the gene luxi in G.xylinus by codon optimization
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<partinfo>BBa_C0061 AddReview 4</partinfo>
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<I>Username</I>
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Enter the review inofrmation here.
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<partinfo>BBa_C0061 AddReview 5</partinfo>
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<I>UNIPV-Pavia iGEM 2011</I>
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<em>
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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>.
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</em>
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<br><br>
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The LuxI enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity, with these parts:
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<ul>
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<li> <A HREF="https://parts.igem.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li>
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<li> <A HREF="https://parts.igem.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li>
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<li> <A HREF="https://parts.igem.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li>
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<li> <A HREF="https://parts.igem.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li></ul>
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<br>
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<em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="https://parts.igem.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator.</em>
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<br><br>
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The four measurment parts <a href="http://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI">p<sub>tet</sub>-RBSx-LuxI</a> were quantitatively characetrized using the BBa_T9002 biosensor.<br>
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The HSL synthesis rate has been evaluated, estimating V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> parameters of the equations below:<br>
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;">
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<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg">
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<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div>
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<br>
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A significative HSL production was observed for <A HREF="https://parts.igem.org/wiki/index.php/Part:BBa_K516210">BBa_K516210</a>, <A HREF="https://parts.igem.org/wiki/index.php/Part:BBa_K516211">BBa_K516211</a> and <A HREF="https://parts.igem.org/wiki/index.php/Part:BBa_K516214">BBa_K516214</a>, while no HSL production was observed for <A HREF="https://parts.igem.orgwiki/index.php/Part:BBa_K516212">BBa_K516212</a>. The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected (HSL concentration measured in the supernatants of cultures, see <a href="https://parts.igem.org/Part:BBa_T9002:Experience#t9002PV11">BBa_T9002 Experience page</a>):
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<div align="justify"><div class="thumbinner" style="width: 850px;">
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<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg">
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<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div>
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<br>
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Parameters' values are summarized in the table below:
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<br>
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<div align="center">
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<table align="center" class='data' border="1">
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<tr>
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<td class='row'><b>V<sub>max</sub></b></td>
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<td class='row'><b>k<sub>M,LuxI</sub></b></td>
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<td class='row'><b>&alpha;<sub>B0030</sub></b></td>
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<td class='row'><b>&alpha;<sub>B0031</sub></b></td>
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<td class='row'><b>&alpha;<sub>B0032</sub></b></td>
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<td class='row'><b>&alpha;<sub>B0034</sub></b></td>
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</tr>
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<tr><td class='row'>3.56*10<sup>-9</sup></td>
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<td class='row'>6.87*10<sup>3</sup></td>
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<td class='row'>87</td>
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<td class='row'>8.5</td>
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<td class='row'>ND</td>
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<td class='row'>252</td>
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</tr></table>
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</div>
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<br>
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The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation.
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<partinfo>BBa_C0061 AddReview 4</partinfo>
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<I>Kevin (iGEM Braunschweig 2013)</I>
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The plasmid pSB1C3 BBa_C0061 from the 2013 distribution Kit was transformed in <I>E. coli</I> XL1 BlueMRF. Sequencing with standard verification primers VR and VF2 showed matching sequence of the pre- and suffix and the part itself but the backbone DNA in front of the prefix does not match pSB1C3.
 
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Latest revision as of 20:18, 1 November 2017

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

Applications of BBa_C0061

User Reviews

UNIQ0ff7176dd2ebd2da-partinfo-00000000-QINU

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

•••••

wmholtz

Using this part, I have successfully constructed and tested a quorum sensing circuit in E. coli.

•••••

Youri

This part was used as a subpart in K546005, K546546 and found to be functional.

;

UNIQ0ff7176dd2ebd2da-partinfo-00000004-QINU 


https://parts.igem.org/Part:BBa_K2267027We improved the gene luxi in G.xylinus by codon optimization

UNIQ0ff7176dd2ebd2da-partinfo-00000005-QINU

•••••

UNIPV-Pavia iGEM 2011

NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C in low copy plasmid pSB4C5.

The LuxI enzyme activity has been characterized under the regulation of ptet promoter, assaying its enzymatic activity, with these parts:


Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid pSB4C5, that contains the BBa_B0054 terminator.

The four measurment parts ptet-RBSx-LuxI were quantitatively characetrized using the BBa_T9002 biosensor.
The HSL synthesis rate has been evaluated, estimating Vmax, kM,LuxI and αRBSx parameters of the equations below:

A significative HSL production was observed for BBa_K516210, BBa_K516211 and BBa_K516214, while no HSL production was observed for BBa_K516212. The parameters Vmax, kM,LuxI and αRBSx were estimated with a simultaneous fitting of the data collected (HSL concentration measured in the supernatants of cultures, see BBa_T9002 Experience page):
Parameters' values are summarized in the table below:
Vmax kM,LuxI αB0030 αB0031 αB0032 αB0034
3.56*10-9 6.87*103 87 8.5 ND 252

The provided parameters kM and Vmax represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation.

••••

Kevin (iGEM Braunschweig 2013)

The plasmid pSB1C3 BBa_C0061 from the 2013 distribution Kit was transformed in E. coli XL1 BlueMRF. Sequencing with standard verification primers VR and VF2 showed matching sequence of the pre- and suffix and the part itself but the backbone DNA in front of the prefix does not match pSB1C3.

;

UNIQ0ff7176dd2ebd2da-partinfo-00000009-QINU