Difference between revisions of "Team:NUS Singapore-A/Demonstrate"

 
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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<img src="https://static.igem.org/mediawiki/2018/9/95/T--NUS_Singapore-A--Demonstrate_heading_C.png" class="header" alt="demonstrate header">
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<button id="OVERVIEW" class="accordion"> THE DEMONSTRATION: AN OUTLINE </button>
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<p>We have developed a novel multicomponent biomanufacturing platform, <i>Coup Dy’état</i>, which serves to facilitate the optimization of biomanufacturing. From the various features that distinguish our system, to the heterologous production of compounds, we have successfully demonstrated that each part of our system works as intended. We have also shown how several of the components have been integrated into the biomanufacturing process.
  
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<p>
 
 
<h2>XYLOSE AS FEEDSTOCK</h2>
 
<h2>XYLOSE AS FEEDSTOCK</h2>
  
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<p>✔Successfully constructed an inducible xylose-utilizing module</p>
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<p>✔ Successfully constructed an inducible xylose-utilizing module</p>
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<p>✔Demonstrated improved growth of <i>E. coli</i> BL21* containing this module in xylose and glucose-xylose mixture</p>
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<p>✔ <b>Demonstrated improved growth of <i>E. coli</i> BL21* containing this module in xylose and glucose-xylose mixture</b></p>
  
 
<h2>DE NOVO BIOSYNTHESIS</h2>
 
<h2>DE NOVO BIOSYNTHESIS</h2>
  
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<p>✔Successfully constructed a naringenin-producing plasmid with just a single missing enzyme required for full de novo synthesis</p>
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<p>✔ Successfully constructed a naringenin-producing plasmid (with just a single missing enzyme) required for full de novo synthesis</p>
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<p>✔Demonstrated production of naringenin from coumaric acid in E. coli Acella and BL21*</p>
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<p>✔ <b>Demonstrated the production of naringenin from coumaric acid in <i>E. coli</i> Acella and BL21*</b></p>
  
 
<h2>LUTEOLIN</h2>
 
<h2>LUTEOLIN</h2>
  
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<p>✔Successfully constructed chemically-inducible and light inducible luteolin-producing plasmids</p>
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<p>✔ Successfully constructed chemically-inducible and light inducible luteolin-producing plasmids</p>
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<p>✔Demonstrated production of luteolin from naringenin in E. coli BL21*</p>
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<p>✔ Characterized expression of flavonoid 3′-hydroxylase (F3′H) under EL222 blue light repressible promoter P<sub>BLrep</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2819200" target="_blank">Bba_K2819200</a>)</p>
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<p>✔Characterized Bba_ (F3’H), Bba (FNS), Bba (pBAD-FNS)</p>
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<p>✔ Characterized expression of flavonoid 3′-hydroxylase (FNS) under arabinose-inducible promoter P<sub>BAD</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2819206" target="_blank">Bba_K2819206)</a></p>
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<p>✔ <b>Demonstrated the production and extraction of luteolin from naringenin in <i>E. coli</i> BL21*, using shake-flask and bioreactor synthesis</b> (see <a href="#LBS">below</a>)</p>
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<h2>BLUE LIGHT REPRESSIBLE SYSTEM</h2>
 
<h2>BLUE LIGHT REPRESSIBLE SYSTEM</h2>
  
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<p>✔Improved characterization of EL222 blue light repressible promoter, P<sub>BLrep</sub> (BBa_)</p>
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<p>✔ Improved characterization of EL222 blue light repressible promoter P<sub>BLrep</sub> (<a href="http://parts.igem.org/Part:BBa_K2819103" target="_blank">BBa_K2819103</a>)</p>
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<p>✔Improved characterization of EL222 blue light inducible promoter, P<sub>BLind</sub> (BBa_)</p>
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<p>✔ <b>Demonstrated blue light repressible control of luteolin production</b></p>
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<p>✔Demonstrated blue light repression of luteolin production</p>
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<h2>STRESS REPORTER</h2>  
 
<h2>STRESS REPORTER</h2>  
  
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<p>✔Successfully constructed a stress reporter module</p>
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<p>✔ Successfully constructed a stress reporter module</p>
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<p>✔Characterized the burden responsive promoter, P<sub>htpG1</sub></p>
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<p>✔ Characterized the burden-responsive promoter P<sub>htpG1</sub> (<a href="http://parts.igem.org/Part:BBa_K2819010" target="_blank">BBa_K2819010</a>)</p>
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<p>✔Demonstrated stress induced by naringenin- and luteolin-producing plasmids</p>
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<p>✔ Demonstrated that reporter is robust under across different genetic backgrounds and temperatures</p>
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<p>✔ <b>Demonstrated that stress was induced by naringenin- and luteolin-producing plasmids, which was detected and reported by the stress reporter module</b></p>
  
 
<h2>CELL-MACHINE INTERFACE</h2>
 
<h2>CELL-MACHINE INTERFACE</h2>
  
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<p>✔Designed and built devices which help characterize optogenetic circuits in petri dishes and 250 ml conical flasks</p>
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<p>✔ Designed and built devices which help characterize optogenetic circuits in petri dishes and 250 ml conical flasks</p>
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<p>✔Designed and built a 500 ml working volume benchtop optogenetic bioreactor, which comprises a peristaltic pump, 2-in-1 OD and fluorescence sensor, and fermentation chamber</p>
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<p>✔ Designed and built a 500 ml working volume benchtop optogenetic bioreactor, which comprises a peristaltic pump, 2-in-1 OD and fluorescence sensor, and fermentation chamber</p>
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<p>✔Designed and implemented a feedback control system to control the optogenetic bioreactor</p>
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<p>✔ Designed and implemented a feedback control system to control the optogenetic bioreactor</p>
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<p>✔ Demonstrated the ability of 2-in-1 OD and fluorescence sensor to measure OD<sub>600</sub> and fluorescence</p>
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<p>✔ <b>Demonstrated the ability of the feedback control system to turn off blue light for protein production under P<sub>BLrep</sub> when OD<sub>600</sub> reaches 0.600</b></p>
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<button id="LBS" class="accordion"> LUTEOLIN SYNTHESIS IN BIOREACTOR </button>
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
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  To bring ourselves one step closer to actual bioproduction, we scaled up our shake-flask biosynthesis to a 2 L bioreactor. After harvesting the sample, we carried out our safety protocol (see: <a href="https://2018.igem.org/Team:NUS_Singapore-A/Safety"><i>Safety</i></a>) and also attempted to extract our target compound, luteolin.
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<figure id="DemoImg01" class="figures2">
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  <img src="https://static.igem.org/mediawiki/2018/6/6d/T--NUS_Singapore-A--Demo_biosynthesis.jpg" alt="Demo Image 04">
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    <figcaption style="text-align:center">Start of reaction</figcaption>
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  <figure id="DemoImg03" class="figures2">
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  <img src="https://static.igem.org/mediawiki/2018/3/3c/T--NUS_Singapore-A--Demo_extraction.jpg" alt="Demo Image 03">
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    <figcaption>Process of extraction</figcaption>
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    <img src="https://static.igem.org/mediawiki/2018/4/46/T--NUS_Singapore-A--IHP_LeongMinyi_Holding_Luteolin.jpeg">
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      <figcaption>Fashion designer-approved!</figcaption>
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  <video src="https://static.igem.org/mediawiki/2018/e/ee/T--NUS_Singapore-A--Results_Luteolin_Bioreactor.mp4" type="video/mp4" controls Autoplay=autoplay></video>
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      <figcaption style="text-align:center;"><i>Bioreactor in action!</i></figcaption>
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  <img src="https://static.igem.org/mediawiki/2018/6/6b/T--NUS_Singapore-A--Demo_YellowHarvest.jpg" alt="Demo Image 02">
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    <figcaption style="text-align:center">Harvested sample</figcaption>
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  <figure id="DemoImg04" class="figures2">
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  <img src="https://static.igem.org/mediawiki/2018/e/e5/T--NUS_Singapore-A--Demo_ExtractedDyes.jpg" alt="Demo Image 04">
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    <figcaption style="text-align:center">The extracted product</figcaption>
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<p>Upon completion of the whole biomanufacturing process, we brought the fruits of our labour back to Ms Leong Minyi, a fashion designer who was a chief source of inspiration for <i>Coup Dy'état</i>, thus coming full circle for our project  (see: <a href="https://2018.igem.org/Team:NUS_Singapore-A/Human_Practices#iHP"><i>Integrated Human Practices</i></a>).
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{{:Team:NUS_Singapore-A/Templates/Footer}}

Latest revision as of 21:11, 17 October 2018

CONNECT WITH US
demonstrate header

We have developed a novel multicomponent biomanufacturing platform, Coup Dy’état, which serves to facilitate the optimization of biomanufacturing. From the various features that distinguish our system, to the heterologous production of compounds, we have successfully demonstrated that each part of our system works as intended. We have also shown how several of the components have been integrated into the biomanufacturing process.

XYLOSE AS FEEDSTOCK

✔ Successfully constructed an inducible xylose-utilizing module

✔ Demonstrated improved growth of E. coli BL21* containing this module in xylose and glucose-xylose mixture

DE NOVO BIOSYNTHESIS

✔ Successfully constructed a naringenin-producing plasmid (with just a single missing enzyme) required for full de novo synthesis

✔ Demonstrated the production of naringenin from coumaric acid in E. coli Acella and BL21*

LUTEOLIN

✔ Successfully constructed chemically-inducible and light inducible luteolin-producing plasmids

✔ Characterized expression of flavonoid 3′-hydroxylase (F3′H) under EL222 blue light repressible promoter PBLrep (Bba_K2819200)

✔ Characterized expression of flavonoid 3′-hydroxylase (FNS) under arabinose-inducible promoter PBAD (Bba_K2819206)

✔ Demonstrated the production and extraction of luteolin from naringenin in E. coli BL21*, using shake-flask and bioreactor synthesis (see below)

BLUE LIGHT REPRESSIBLE SYSTEM

✔ Improved characterization of EL222 blue light repressible promoter PBLrep (BBa_K2819103)

✔ Demonstrated blue light repressible control of luteolin production

STRESS REPORTER

✔ Successfully constructed a stress reporter module

✔ Characterized the burden-responsive promoter PhtpG1 (BBa_K2819010)

✔ Demonstrated that reporter is robust under across different genetic backgrounds and temperatures

✔ Demonstrated that stress was induced by naringenin- and luteolin-producing plasmids, which was detected and reported by the stress reporter module

CELL-MACHINE INTERFACE

✔ Designed and built devices which help characterize optogenetic circuits in petri dishes and 250 ml conical flasks

✔ Designed and built a 500 ml working volume benchtop optogenetic bioreactor, which comprises a peristaltic pump, 2-in-1 OD and fluorescence sensor, and fermentation chamber

✔ Designed and implemented a feedback control system to control the optogenetic bioreactor

✔ Demonstrated the ability of 2-in-1 OD and fluorescence sensor to measure OD600 and fluorescence

✔ Demonstrated the ability of the feedback control system to turn off blue light for protein production under PBLrep when OD600 reaches 0.600





To bring ourselves one step closer to actual bioproduction, we scaled up our shake-flask biosynthesis to a 2 L bioreactor. After harvesting the sample, we carried out our safety protocol (see: Safety) and also attempted to extract our target compound, luteolin.


Demo Image 04
Start of reaction

Demo Image 03
Process of extraction

Fashion designer-approved!

Bioreactor in action!

Demo Image 02
Harvested sample

Demo Image 04
The extracted product


Upon completion of the whole biomanufacturing process, we brought the fruits of our labour back to Ms Leong Minyi, a fashion designer who was a chief source of inspiration for Coup Dy'état, thus coming full circle for our project (see: Integrated Human Practices).









HEAR OUR STORY

We’re using synthetic biology to change the way the world thinks and functions.

We’re a little disruptive, dangerous and maybe a tad too bold. But the world needs some shaking up every now and then, and we think we’re just the right people for that.

Together we’re going to engineer biology and create something awesome.

CONTACT US

nusigem@gmail.com

21 Lower Kent Ridge Rd, Singapore 119077