Team:Peking/DataPage

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<h3 id=”tilte1”>Data for favorite parts</h3>
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<h3 id="title2">Data for Favorite Parts</h3>
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<b>Optimized Luminesensor(BBa_K819005)</b> a fusion protein consisting of an <i>E. coli</i> endogenous SOS system repressor LexA, carrying mutations at positions 40-42, and fungus <i>N. crass</i> photosensor protein VVD, carrying mutations N56K, C71V and I135M. This part serves as an ultra-sensitive photoreceptor which can sense light as weak as moonlight and will induce light-dependent repression of genes with their promoters containing mutated 408 forms of the SOS box.<br /><br /><b>Testing device for Luminesensor(BBa_K819006) and measurement device for Luminesensor(BBa_K819007)</b> a fast degrading GFP ligated to the downstream of SulA and RecA promoters, both in 408 form (only recognizable by our LexA-VVD fusing protein, not by E.coli endogenous LexA). The SulA promoter, which belongs to the SOS regulon family, promotes the expression of SulA protein gene which expresses SulA protein, a cell division inhibitor. The RecA promoter promotes the expression of RecA protein, which cuts the dimerized LexA protein in response to double strand break. When <i>E. coli</i> cells co-transformed with our luminesensor plasmid and light-responsive reporter plasmids are illuminated by blue light, the LexA408-VVD fusion protein expressed within the cell will dimerize in response to the light. The dimerized LexA408 domains will bind the SOS box (in 408 form) of both the SulA408 promoter and RecA408 promoter to inhibit the transcription of the downstream gene so that no GFP will be expressed. If the surrounding environment is dark, the luminesensor will not dimerize and no supression of the promoters will occur, leading to the expression of GFP.
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An <b>Constitutive LuxBrick Generator(<a href="http://partsregistry.org/Part:BBa_K819005">Main Page-BBa_K819005</a>)</b>  
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<br /><br /><b>Testing device for the <b><i>Luminesensor</i></b> (<a href="http://partsregistry.org/Part:BBa_K819006">Main Page-BBa_K819006</a>) and measurement device for the Luminesensor (<a href="http://partsregistry.org/Part:BBa_K819007">Main Page-BBa_K819007</a>)</b> a fast degrading GFP ligated to the downstream of SulA408 promoter and RecA408 promoter, respectively. These two promoters are regulated by the <i>Luminesensor</i> and works independently of the host genetic context.
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<h3 id=”tilte2”>Data for optimized parts</h3>
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<h3 id="title3">Data for Optimized Parts</h3>
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  this comes later.
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  <img src="/wiki/images/1/1c/Peking2012_part_T7_operon.png" alt="Fig 1. BL21 cells harboring T7-operon(BBa_K819008) are emitting blue luminescence under IPTG induction" style="width:175px;" />
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  <p class="description" style="width:265px;">Figure 2. BL21 cells harboring T7-lux operon(BBa_K819008) are emitting blue luminescence under IPTG induction at 5x10<sup>-5</sup>M</p>
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<p><b>Lux Operon under T7 promoter (<a href="http://partsregistry.org/Part:BBa_K819008">Main Page-BBa_K819008</a>)</b><br/>We successfully improved the easy-of-use of “lux brick” (<a href="http://partsregistry.org/Part:BBa_K325909">BBa_K325909</a>) part constructed by Cambridge 2010 iGEM team by placing the coding sequence of luxbrick (luxCDABEG) and related RBS under T7 promoter (<a href="http://partsregistry.org/Part:BBa_I712074">BBa_I712074</a>).
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<br/><br/> T7 promoter separates sensing/circuitry functions from pathways/actuation. It is encoded in genetically distinct regions while linked with other circuits by providing the output for the circuits that drive the expression of phage polymerases. Luxbrick under T7 promoter is very modular, because it is transcribed by T7 polymerase, which can be placed under any other promoter, forming a interface between luxbrick and other systems.
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Once transformed into BL21(de3)strain, high protein production can be achieved rapidly after IPTG induction.Final concentration of IPTG should be round 0.05mM. <br />
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<br/><br/> To note that the incubating temperature should be no higher than 30<sup>o</sup>C, or the heavy Lux complex can easily aggregate.
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Optimum incubating conditions provided by Peking iGEM 2012:
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  250 rpm, 22<sup>o</sup>C, good ventilation after induction(final concentration of IPTG: round 0.5 mM).
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<h3 id=”tilte3”>Data for other submitted parts</h3>
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<h3 id="title4">Data for Other Submitted Parts</h3>
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this also comes later.
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<b><i>Luminesensor</i> repressible SulA408 promoter (<a href="http://partsregistry.org/Part:BBa_K819017">Main Page-BBa_K819017</a>) <br/><i>Luminesensor</i> repressible RecA408 promoter (<a href="http://partsregistry.org/Part:BBa_K819002">Main Page-BBa_K819002</a>)</b><br />These are the two promoters used in this year’s project to test the functionality of <b><i>Luminesensor</i></b> and measure the efficiency of it.
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<br /><br /><b>Weak CheZ generator (<a href="http://partsregistry.org/Part:BBa_K819009">Main Page-BBa_K819009</a>)<br/>
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medium CheZ generator (<a href="http://partsregistry.org/Part:BBa_K819010">Main Page-BBa_K819010</a>)</b><br />Chemotaxis protein CheZ ligated to 2 constitutive promoters with varying strengths. These two parts express the CheZ protein in varied quantities, which corresponds to the different levels of induced mobility in the &Delta;CheZ <i>E.coli</i> strain.
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<br /><br /><b><i>Luminesensor</i> repressible RecA408 promoter (<a href="http://partsregistry.org/Part:BBa_K819015">Main Page-BBa_K819015</a>)<br/><i>Luminesensor</i> repressible SulA408 promoter (<a href="http://partsregistry.org/Part:BBa_K819016">Main Page-BBa_K819016</a>)</b><br />Chemotaxis protein CheZ ligated to the <b><i>Luminesensor</i></b> repressible SulA408 promoter and RecA408 promoter, respectively. Presumably these two parts can would convert chemotaxis pathway of <i>E.coli</i> into a phototaxis pathway by controlling the expression level of CheZ in &Delta;CheZ strain in response to blue light.
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<br /><br /><b><i>Luminesensor</i> repressible LacIM generator</i> (<a href="http://partsregistry.org/Part:BBa_K819012">Main Page-BBa_K819012</a>)</b><br />Coding sequence of LacI mutant protein driven by the <b><i>Luminesensor</i></b> repressible SulA408 promoter (<a href="http://partsregistry.org/Part:BBa_K819017">BBa_K819017</a>).
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  <h3 id=”tilte4”>Data for existing parts</h3>
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  <h3 id="title5">Data for Pre-Existing Parts</h3>
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  <p><b>luxbrick (<a href="http://partsregistry.org/Part:BBa_K325909">Main Page-BBa_K325909</a>)</b> <br/>luxbrick is a bacteria luciferase part constructed by Cambridge 2010 iGEM team. They successfully expressed bacterial luciferase in E.coli, which could produce blue light under L-arabinose induction, and they carefully characterized this part. But because there are data we need while they didn’t give, we made the following supplementary characterization.<br /><br />The supplementary characterization and our application of luxbrick part were added to the experience page of this part (see <a href="http://partsregistry.org/Part:BBa_K325909:Experience">Experience-BBa_K325909</a>)<br /><br />The spectrum of the light emitted from <i>E.coli</i> harboring luxbrick was measured and it showed a maximum intensity at 485nm.</p>
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  <img src="/wiki/images/c/c3/Peking2012_light_communication_3.png" alt="Figure 3" />
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  <p class="description">Figure 3. TOP10 cells harboring the luxbrick were cultured in LB medium and induced with L-arabinose at 10<sup>-3</sup>M. 10 hours after induction, the glowing cells were measured for spectrum using SHIMADZU RF5301PC Spectrofluorophotometer.</p>
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<p>The time course of luxbrick expression and blue light emission under L-arabinose induction was given in a more visualized way, which is a short movie. As shown in the movie, the cells transformed with luxbrick begin to glow 9 hours after induction, and the entire visible glowing process lasts about 10 hours.</p>
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<br />if you can not view this video，please click <a href="http://v.youku.com/v_show/id_XNDU0NDU0NDcy.html">HERE</a>.
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  <p class="description">Movie1. the time course of our light emitting cell</p>
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</html><groupparts>iGEM012 Peking</groupparts><html></div></html>{{Template:Peking2012_Color_Epilogue}}
</html><groupparts>iGEM012 Peking</groupparts><html></div></html>{{Template:Peking2012_Color_Epilogue}}

Latest revision as of 14:35, 20 October 2012

Data for Favorite Parts

An Constitutive LuxBrick Generator(Main Page-BBa_K819005)

Testing device for the Luminesensor (Main Page-BBa_K819006) and measurement device for the Luminesensor (Main Page-BBa_K819007) a fast degrading GFP ligated to the downstream of SulA408 promoter and RecA408 promoter, respectively. These two promoters are regulated by the Luminesensor and works independently of the host genetic context.

Data for Optimized Parts

Fig 1. BL21 cells harboring T7-operon(BBa_K819008) are emitting blue luminescence under IPTG induction

Figure 2. BL21 cells harboring T7-lux operon(BBa_K819008) are emitting blue luminescence under IPTG induction at 5x10-5M

Lux Operon under T7 promoter (Main Page-BBa_K819008)
We successfully improved the easy-of-use of “lux brick” (BBa_K325909) part constructed by Cambridge 2010 iGEM team by placing the coding sequence of luxbrick (luxCDABEG) and related RBS under T7 promoter (BBa_I712074).

T7 promoter separates sensing/circuitry functions from pathways/actuation. It is encoded in genetically distinct regions while linked with other circuits by providing the output for the circuits that drive the expression of phage polymerases. Luxbrick under T7 promoter is very modular, because it is transcribed by T7 polymerase, which can be placed under any other promoter, forming a interface between luxbrick and other systems. Once transformed into BL21(de3)strain, high protein production can be achieved rapidly after IPTG induction.Final concentration of IPTG should be round 0.05mM.


To note that the incubating temperature should be no higher than 30oC, or the heavy Lux complex can easily aggregate. Optimum incubating conditions provided by Peking iGEM 2012: 250 rpm, 22oC, good ventilation after induction(final concentration of IPTG: round 0.5 mM).

Data for Other Submitted Parts

Luminesensor repressible SulA408 promoter (Main Page-BBa_K819017)
Luminesensor repressible RecA408 promoter (Main Page-BBa_K819002)
These are the two promoters used in this year’s project to test the functionality of Luminesensor and measure the efficiency of it.

Weak CheZ generator (Main Page-BBa_K819009)
medium CheZ generator (Main Page-BBa_K819010)
Chemotaxis protein CheZ ligated to 2 constitutive promoters with varying strengths. These two parts express the CheZ protein in varied quantities, which corresponds to the different levels of induced mobility in the ΔCheZ E.coli strain.

Luminesensor repressible RecA408 promoter (Main Page-BBa_K819015)
Luminesensor repressible SulA408 promoter (Main Page-BBa_K819016)
Chemotaxis protein CheZ ligated to the Luminesensor repressible SulA408 promoter and RecA408 promoter, respectively. Presumably these two parts can would convert chemotaxis pathway of E.coli into a phototaxis pathway by controlling the expression level of CheZ in ΔCheZ strain in response to blue light.

Luminesensor repressible LacIM generator (Main Page-BBa_K819012)
Coding sequence of LacI mutant protein driven by the Luminesensor repressible SulA408 promoter (BBa_K819017).

Data for Pre-Existing Parts

luxbrick (Main Page-BBa_K325909)
luxbrick is a bacteria luciferase part constructed by Cambridge 2010 iGEM team. They successfully expressed bacterial luciferase in E.coli, which could produce blue light under L-arabinose induction, and they carefully characterized this part. But because there are data we need while they didn’t give, we made the following supplementary characterization.

The supplementary characterization and our application of luxbrick part were added to the experience page of this part (see Experience-BBa_K325909)

The spectrum of the light emitted from E.coli harboring luxbrick was measured and it showed a maximum intensity at 485nm.

Figure 3

Figure 3. TOP10 cells harboring the luxbrick were cultured in LB medium and induced with L-arabinose at 10-3M. 10 hours after induction, the glowing cells were measured for spectrum using SHIMADZU RF5301PC Spectrofluorophotometer.

The time course of luxbrick expression and blue light emission under L-arabinose induction was given in a more visualized way, which is a short movie. As shown in the movie, the cells transformed with luxbrick begin to glow 9 hours after induction, and the entire visible glowing process lasts about 10 hours.


if you can not view this video，please click HERE.

Movie1. the time course of our light emitting cell

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