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

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        <h1><i>Coup Dy’état</i></h1>
 +
        <br>
 +
        <p> A <i>coup d’état</i> is the sudden overthrow of a government through illegal force by a small group.</p>
 +
        <br>
 +
        <p> It is in this spirit that we offer <i>Coup Dy’état</i>, our synthetic biology solution.</p>
 +
 +
        <h2><b>Eco-friendly Biomanufacturing of Flavonoid Dyes in <i>Escherichia coli</i> via Computer-mediated Optogenetic Regulation</h2>
 +
        </p>
 +
        <br>
 +
        <br>
 +
        <hr>
 +
        <br>
 +
 +
      <h2>The Problem</h2>
 +
      <div class="row">
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        <div class="column left">
 +
          <p>Water pollution is a key problem in the textile dyeing and dyestuff production industry. In many regions of the world, rivers and water bodies that are sources of drinking water and livelihood are becoming heavily polluted by textile dyeing.
 +
            This is largely due to the <b>irresponsible disposal</b> of <b>industrial effluents</b>, <b>hard-to-biodegrade synthetic dyes</b> by <b>textiles and dyestuff producers</b> and <b>ineffective wastewater treatment</b>.
 +
          </p>
 +
          <br>
 +
          <figure class="figures">
 +
            <img src="https://www.straitstimes.com/sites/default/files/articles/2018/01/10/ST_20180110_WYCITARUM10_3678367.jpg">
 +
            <figcaption><b><i>Image 1. Citarum River. A villager navigates his way through heavily dye-polluted waters. Photo: Iqbal Ksumadireza, The Straits Times,
 +
2018 </i></b>
 +
            </figcaption>
 +
          </figure>
 +
          <br>
 +
          <h3>What is Happening</h3>
 +
          <p>Already, the Citarum River in Indonesia is a clear example of this murky problem. The <b>30 million residents</b> relying on the river as their only water source and livelihood are experiencing adverse skin conditions and increased exposure
 +
            to infectious diseases<sup>[1]</sup>, while the river has almost no aquatic life left<sup>[2]</sup>. According to a 2015 UN report, in some areas of the river, lead levels at more than <b>1,000 times the USEPA standard</b> in drinking water
 +
            have been found<sup>[3]</sup>.
 +
          </p>
 +
          <br>
 +
          <figure class="figures">
 +
            <img src="https://static.igem.org/mediawiki/2018/6/6f/T--NUS_Singapore-A--KC_Hardship.png">
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            <figcaption><b>Image 2</b>. "In some areas of the river, lead levels at more than 1,000 times the USEPA standard in drinking water."
 +
            </figcaption>
 +
          </figure>
 +
          <br>
 +
        </div>
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        <div class="column right">
 +
          <p>River water rapidly changing colours from red, to green, yellow, and black due to high concentrations of dye is not an uncommon sight. However, Citarum River is not an isolated case. Similar sights can be seen
 +
            in Pearl River in China, Buriganga River in Bangladesh, and Bagmati River in India. In Dhaka, Bangladesh, <b>719 factories and textile mills</b> generate close to <b>200 metric tons of wastewater per ton of fabric per year</b><sup>[4]</sup>. According to the World Bank, on the global scale, the textile dyeing and dyestuff production industry is the <b>second most pollutive industry</b>, coming in only after oil, and produces <b>a fifth of the world’s water pollution</b>. It also <b>uses the most water</b> apart from agriculture<sup>[5]</sup>.
 +
          </p>
 +
          <br>
 +
          <figure class="figures">
 +
            <img src="https://static.igem.org/mediawiki/2018/0/01/T--NUS_Singapore-A--KC_Pollution.png">
 +
            <figcaption><b>Image 3</b>. "...textile dyeing and dyestuff production industry is the second most pollutive industry."
 +
            </figcaption>
 +
          </figure>
 +
          <br>
 +
          <p>
 +
            Addressing this problem through legislation has not been effective and treatment of the waste is expensive. In recent years, some efforts have been put in to <b>revive natural dyes</b> as a more sustainable alternative to the synthetic dyes<sup>[6]</sup>. However, this motion has not been gaining much traction, as traditional natural dye production which extracts dye from plants faces many constraints. Natural dyes produced traditionally often have <b>inconsistent quality</b>, varying from batch to batch of plants used. Furthermore, it is <b>land and labour intensive</b> and <b>competes with food production</b> for land use.
 +
          </p>
 +
          <br>
 +
          <figure class="figures">
 +
            <img src="https://static.igem.org/mediawiki/2018/8/84/T--NUS_Singapore-A--KC_NatDyes.png">
 +
            <figcaption><b>Image 4</b>. "...it (natural dye production) is land and labour intensive and competes with food production for land use."
 +
            </figcaption>
 +
          </figure>
 +
          <br>
 +
        </div>
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      </div>
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      <br>
 +
      <hr>
 +
      <br>
 +
 +
      <div class="row">
 +
        <div class="column left">
 +
          <h2>Our Motivation</h2>
 +
          <br>
 +
          <p>Our team strongly believes that the current approach of producing synthetic dyes is unsustainable. The world urgently needs a more sustainable and eco-friendly solution. To this end, we have taken the <b>synthetic biology approach to natural dye bioproduction</b> in the hopes of making natural dyes a better substitute for synthetic dyes. What this means is our dyes have to be <b>non-toxic</b>, have <b>reduced use of chemicals in its production</b>, <b>environmentally friendly</b> and still remain <b>appealing to fashion designers and consumers</b>.
 +
          </p>
 +
          <br>
 +
          <p>
 +
            Our project entitled <b>Coup Dy'état</b> aims to develop a new bio-manufacturing method of producing flavonoids in <i>E. coli</i> for use as natural dyes.
 +
          </p>
 +
          <br>
 +
          <figure class="figures">
 +
            <img src="https://static.igem.org/mediawiki/2018/9/94/T--NUS_Singapore-A--KC_Consumers.png" style="width:32%;">
 +
            <img src="https://static.igem.org/mediawiki/2018/0/08/T--NUS_Singapore-A--KC_Application.png"  style="width:32%;">
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            <img src="https://static.igem.org/mediawiki/2018/c/cf/T--NUS_Singapore-A--KC_Experimentation.png"  style="width:32%;">
 +
            <figcaption><b>Image 5</b>. Our dyes be non-toxic, have reduced use of chemicals in its production, environmentally friendly and still remain <b>appealing to fashion designers and consumers
 +
            </figcaption>
 +
          </figure>
 +
          <br>
 +
        </div>
 +
        <div class="column right">
 +
          <h2>The Challenges</h2>
 +
          <br>
 +
          <p>However, in current bio-manufacturing methods, constraints and limitations prevent bio-manufacturing from achieving more of its potential. One limitation is <b>cost</b>, as <b>expensive chemical inducers</b> and <b>feedstock</b> are often required
 +
            during the bio-manufacturing. Chemical inducers are often used in production to activate gene expression when cells have reached high cell density. The feedstock can make up as much as <b>18% of total production costs</b><sup>[7]</sup>.
 +
          </p>
 +
          <br>
 +
          <p>
 +
            Although environmental stresses are currently taken into consideration and managed in bioreactors, there is <b>one form of cell stress that is often neglected</b>. <b>Stress induced by the expression of recombinant proteins </b> is present
 +
            not only in large-scale bioreactor, but also in bench-top laboratory experiments. The depletion of finite cellular resources during the expression of synthetic constructs constitutes an unwanted burden, hampering the growth and expected performance
 +
            of engineered cells in an unpredictable manner<sup>[8]</sup>. The bio-manufacturing process can consume as much as 25% of all cellular transcriptional resources<sup>[9]</sup>. Unfortunately, there is a lack of monitoring for such cell stress
 +
            within industrial bioreactors, leading to cell death that is counterproductive to bio-manufacturing.
 +
          </p>
 +
          <br>
 +
        </div>
 +
      </div>
 +
      <br>
 +
      <hr>
 +
      <br>
 +
 +
      <h2>Our Solution</h2>
 +
      <br>
 +
 +
      <figure class="figures">
 +
        <img src="https://static.igem.org/mediawiki/2018/3/34/T--NUS_Singapore-A--Description_overview.png">
 +
        <figcaption><b><i>Figure 1. Solution Overview <br>Top row (from left): Xylose, De Novo Biosynthesis of Naringenin, Luteolin production. <br>Bottom row (from left):Blue light repressible system, RFP cell stress reporter, cell-machine interface </i></b> </figcaption>
 +
      </figure>
 +
      <br>
 +
      <figure class="figures">
 +
        <img src="https://static.igem.org/mediawiki/2018/c/ce/T--NUS_Singapore-A--Description_illustration.png">
 +
        <figcaption><b><i>Figure 2. Platform solution developed</figcaption>
 +
      </figure>
 +
 +
      <div class="row">
 +
        <div class="column left">
 +
          <br>
 +
          <p>To address the challenges, our team decided to develop a <b>computer-mediated optogenetic regulation system that serves as a platform</b> for future bio-manufacturing and biosynthesis. The system includes <b>an engineered cell in which its biosynthesis
 +
            pathway can be rapidly regulated by light and having burden-based cellular stress monitored and regulated by a specially designed hardware</b>.
 +
          </p>
 +
          <br>
 +
          <p>
 +
            Firstly, to <b>eliminate the use of expensive chemical inducers</b> to switch from growth to the production phase, and we aim to use <b>optogenetic control</b>. <b>To allow dynamic gene regulation in our engineered bacteria, we designed an optogenetic circuit using a blue light repressible promoter for flavonoid biosynthesis.</b>            With proper positioning of blue light LEDs, by switching on and off blue light, we can achieve more precise and effective activating and repressing gene activity. Unlike chemical inducers, light can be easily and quickly removed from the bioreactor
 +
            to allow for de-repression of gene activity.
 +
          </p>
 +
          <br>
 +
          <p>
 +
            We also engineered our cells to <b>promote xylose uptake</b> in order to enable the <b> use of lignocellulosic waste as a much more sustainable feedstock</b> for our cells. Lignocellulosic waste is a renewable source of biomass from which
 +
            xylose can be processed from. The use of xylose over glucose is also advantageous, as metabolism of glucose will result in the production of acetic acid, a growth inhibitor<sup>[10]</sup>. This could cause bio-manufacturing to be less efficient,
 +
            achieve lower yield and make it less economically viable.
 +
          </p>
 +
          <br>
 +
        </div>
 +
        <div class="column right">
 +
          <br>
 +
          <p>
 +
            Secondly, as it is critical to monitor cellular stress induced by the expression of recombinant proteins for efficient production, <b>we introduced a stress-sensing fluorescence reporter</b>. Placed under a burden-responsive promoter, the
 +
            expression of RFP will increase in proportion with cellular burden, giving us the much-needed feedback in order to take corrective actions to reduce cell stress by decreasing the expression of the synthetic construct.
 +
          </p>
 +
          <br>
 +
          <p>
 +
            Lastly, in order to enhance the feedback operation and ensure bio-manufacturing is constantly optimised and at its most optimal state, a <b>computer-aided system</b> was developed to <b>automatically regulate gene expression using light according to the feedback from the stress sensor</b>.
 +
            This creates a <b>closed-loop system</b> that is devoid of manual inputs. The metabolic burden from the expression of recombinant proteins is dynamic and constantly changing, and the regulation of cell stress can only be made possible through the use of a closed-loop feedback system, which is what we have achieved. To demonstrate this approach, we produced Luteolin, a natural yellow dye.
 +
          </p>
 +
          <br>
 +
        </div>
 +
      </div>
 +
 +
<br><h2>References</h2><br>
 +
 +
<div class="reference">
 +
 +
[1]  France-Presse, Agence. Indonesia takes on task of cleaning up 'world's dirtiest river'. 2 March, 2018. 3 July, 2018. https://www.thenational.ae/world/asia/indonesia-takes-on-task-of-cleaning-up-world-s-dirtiest-river-1.709520 <br><br>
 +
[2] Asian Development Bank; The World Bank. Downstream Impacts of Water Pollution in the Upper Citarum River, West Java, Indonesia. Technical Paper. Manila: Asian Development Bank, 2013. Document. https://www.adb.org/sites/default/files/publication/154493/citarum-river-downstream-impacts-water-pollution.pdf<br><br>
 +
[3] Visvanathan, C. "3Rs for Water Security in Asia and the Pacific." United Nations Centre for Regional Development. 2015. 17. Document. http://www.uncrd.or.jp/content/documents/2660Pre-Final-BG-Plenary%20Session-6_FINAL.pdf<br><br>
 +
[4] Chequer, F. M., de Oliveira, G. A., Ferraz, E. R., Cardoso, J. C., Zanoni, M. V., & de Oliveira, D. P. (2013). Textile Dyes: Dyeing Process and Environmental Impact. IntechOpen, 151-176.<br><br>
 +
[5]  Asian Development Bank; The World Bank. (2013). Downstream Impacts of Water Pollution in the Upper Citarum River, West Java, Indonesia. Manila: Asian Development Bank. Retrieved from https://www.adb.org/sites/default/files/publication/154493/citarum-river-downstream-impacts-water-pollution.pdf<br><br>
 +
[6] Křížová, Hana. (2015). Natural dyes: their past, present, future and sustainability. 59-71. <br><br>
 +
[7] Choi, W. (2018, Oct 11). Cost of Feedstock in Bio-based manufacturing. (A. Leow, Interviewer) <br><br>
 +
[8]  Gasser, B., Saloheimo, M., Rinas, U., Dragosits, M., Rodríguez-Carmona, E., Baumann, K., ... & Porro, D. (2008). Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microbial cell factories, 7(1), 11.<br><br>
 +
[9] Ceroni, F., Boo, A., Furini, S., Gorochowski, T. E., Borkowski, O., Ladak, Y. N., ... & Ellis, T. (2018). Burden-driven feedback control of gene expression. Nature methods, 15(5), 387.<br><br>
 +
[10] Bäcklund et al. Microbial Cell Factories 2011, 10:35 http://www.microbialcellfactories.com/content/10/1/35<br><br>
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Revision as of 02:16, 18 October 2018

CONNECT WITH US
Project Description Header

Coup Dy’état


A coup d’état is the sudden overthrow of a government through illegal force by a small group.


It is in this spirit that we offer Coup Dy’état, our synthetic biology solution.

Eco-friendly Biomanufacturing of Flavonoid Dyes in Escherichia coli via Computer-mediated Optogenetic Regulation




The Problem

Water pollution is a key problem in the textile dyeing and dyestuff production industry. In many regions of the world, rivers and water bodies that are sources of drinking water and livelihood are becoming heavily polluted by textile dyeing. This is largely due to the irresponsible disposal of industrial effluents, hard-to-biodegrade synthetic dyes by textiles and dyestuff producers and ineffective wastewater treatment.


Image 1. Citarum River. A villager navigates his way through heavily dye-polluted waters. Photo: Iqbal Ksumadireza, The Straits Times, 2018

What is Happening

Already, the Citarum River in Indonesia is a clear example of this murky problem. The 30 million residents relying on the river as their only water source and livelihood are experiencing adverse skin conditions and increased exposure to infectious diseases[1], while the river has almost no aquatic life left[2]. According to a 2015 UN report, in some areas of the river, lead levels at more than 1,000 times the USEPA standard in drinking water have been found[3].


Image 2. "In some areas of the river, lead levels at more than 1,000 times the USEPA standard in drinking water."

River water rapidly changing colours from red, to green, yellow, and black due to high concentrations of dye is not an uncommon sight. However, Citarum River is not an isolated case. Similar sights can be seen in Pearl River in China, Buriganga River in Bangladesh, and Bagmati River in India. In Dhaka, Bangladesh, 719 factories and textile mills generate close to 200 metric tons of wastewater per ton of fabric per year[4]. According to the World Bank, on the global scale, the textile dyeing and dyestuff production industry is the second most pollutive industry, coming in only after oil, and produces a fifth of the world’s water pollution. It also uses the most water apart from agriculture[5].


Image 3. "...textile dyeing and dyestuff production industry is the second most pollutive industry."

Addressing this problem through legislation has not been effective and treatment of the waste is expensive. In recent years, some efforts have been put in to revive natural dyes as a more sustainable alternative to the synthetic dyes[6]. However, this motion has not been gaining much traction, as traditional natural dye production which extracts dye from plants faces many constraints. Natural dyes produced traditionally often have inconsistent quality, varying from batch to batch of plants used. Furthermore, it is land and labour intensive and competes with food production for land use.


Image 4. "...it (natural dye production) is land and labour intensive and competes with food production for land use."



Our Motivation


Our team strongly believes that the current approach of producing synthetic dyes is unsustainable. The world urgently needs a more sustainable and eco-friendly solution. To this end, we have taken the synthetic biology approach to natural dye bioproduction in the hopes of making natural dyes a better substitute for synthetic dyes. What this means is our dyes have to be non-toxic, have reduced use of chemicals in its production, environmentally friendly and still remain appealing to fashion designers and consumers.


Our project entitled Coup Dy'état aims to develop a new bio-manufacturing method of producing flavonoids in E. coli for use as natural dyes.


Image 5. Our dyes be non-toxic, have reduced use of chemicals in its production, environmentally friendly and still remain appealing to fashion designers and consumers

The Challenges


However, in current bio-manufacturing methods, constraints and limitations prevent bio-manufacturing from achieving more of its potential. One limitation is cost, as expensive chemical inducers and feedstock are often required during the bio-manufacturing. Chemical inducers are often used in production to activate gene expression when cells have reached high cell density. The feedstock can make up as much as 18% of total production costs[7].


Although environmental stresses are currently taken into consideration and managed in bioreactors, there is one form of cell stress that is often neglected. Stress induced by the expression of recombinant proteins is present not only in large-scale bioreactor, but also in bench-top laboratory experiments. The depletion of finite cellular resources during the expression of synthetic constructs constitutes an unwanted burden, hampering the growth and expected performance of engineered cells in an unpredictable manner[8]. The bio-manufacturing process can consume as much as 25% of all cellular transcriptional resources[9]. Unfortunately, there is a lack of monitoring for such cell stress within industrial bioreactors, leading to cell death that is counterproductive to bio-manufacturing.




Our Solution


Figure 1. Solution Overview
Top row (from left): Xylose, De Novo Biosynthesis of Naringenin, Luteolin production.
Bottom row (from left):Blue light repressible system, RFP cell stress reporter, cell-machine interface

Figure 2. Platform solution developed

To address the challenges, our team decided to develop a computer-mediated optogenetic regulation system that serves as a platform for future bio-manufacturing and biosynthesis. The system includes an engineered cell in which its biosynthesis pathway can be rapidly regulated by light and having burden-based cellular stress monitored and regulated by a specially designed hardware.


Firstly, to eliminate the use of expensive chemical inducers to switch from growth to the production phase, and we aim to use optogenetic control. To allow dynamic gene regulation in our engineered bacteria, we designed an optogenetic circuit using a blue light repressible promoter for flavonoid biosynthesis. With proper positioning of blue light LEDs, by switching on and off blue light, we can achieve more precise and effective activating and repressing gene activity. Unlike chemical inducers, light can be easily and quickly removed from the bioreactor to allow for de-repression of gene activity.


We also engineered our cells to promote xylose uptake in order to enable the use of lignocellulosic waste as a much more sustainable feedstock for our cells. Lignocellulosic waste is a renewable source of biomass from which xylose can be processed from. The use of xylose over glucose is also advantageous, as metabolism of glucose will result in the production of acetic acid, a growth inhibitor[10]. This could cause bio-manufacturing to be less efficient, achieve lower yield and make it less economically viable.



Secondly, as it is critical to monitor cellular stress induced by the expression of recombinant proteins for efficient production, we introduced a stress-sensing fluorescence reporter. Placed under a burden-responsive promoter, the expression of RFP will increase in proportion with cellular burden, giving us the much-needed feedback in order to take corrective actions to reduce cell stress by decreasing the expression of the synthetic construct.


Lastly, in order to enhance the feedback operation and ensure bio-manufacturing is constantly optimised and at its most optimal state, a computer-aided system was developed to automatically regulate gene expression using light according to the feedback from the stress sensor. This creates a closed-loop system that is devoid of manual inputs. The metabolic burden from the expression of recombinant proteins is dynamic and constantly changing, and the regulation of cell stress can only be made possible through the use of a closed-loop feedback system, which is what we have achieved. To demonstrate this approach, we produced Luteolin, a natural yellow dye.



References


[1] France-Presse, Agence. Indonesia takes on task of cleaning up 'world's dirtiest river'. 2 March, 2018. 3 July, 2018. https://www.thenational.ae/world/asia/indonesia-takes-on-task-of-cleaning-up-world-s-dirtiest-river-1.709520

[2] Asian Development Bank; The World Bank. Downstream Impacts of Water Pollution in the Upper Citarum River, West Java, Indonesia. Technical Paper. Manila: Asian Development Bank, 2013. Document. https://www.adb.org/sites/default/files/publication/154493/citarum-river-downstream-impacts-water-pollution.pdf

[3] Visvanathan, C. "3Rs for Water Security in Asia and the Pacific." United Nations Centre for Regional Development. 2015. 17. Document. http://www.uncrd.or.jp/content/documents/2660Pre-Final-BG-Plenary%20Session-6_FINAL.pdf

[4] Chequer, F. M., de Oliveira, G. A., Ferraz, E. R., Cardoso, J. C., Zanoni, M. V., & de Oliveira, D. P. (2013). Textile Dyes: Dyeing Process and Environmental Impact. IntechOpen, 151-176.

[5] Asian Development Bank; The World Bank. (2013). Downstream Impacts of Water Pollution in the Upper Citarum River, West Java, Indonesia. Manila: Asian Development Bank. Retrieved from https://www.adb.org/sites/default/files/publication/154493/citarum-river-downstream-impacts-water-pollution.pdf

[6] Křížová, Hana. (2015). Natural dyes: their past, present, future and sustainability. 59-71.

[7] Choi, W. (2018, Oct 11). Cost of Feedstock in Bio-based manufacturing. (A. Leow, Interviewer)

[8] Gasser, B., Saloheimo, M., Rinas, U., Dragosits, M., Rodríguez-Carmona, E., Baumann, K., ... & Porro, D. (2008). Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microbial cell factories, 7(1), 11.

[9] Ceroni, F., Boo, A., Furini, S., Gorochowski, T. E., Borkowski, O., Ladak, Y. N., ... & Ellis, T. (2018). Burden-driven feedback control of gene expression. Nature methods, 15(5), 387.

[10] Bäcklund et al. Microbial Cell Factories 2011, 10:35 http://www.microbialcellfactories.com/content/10/1/35



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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.

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Project Description Header

Coup Dy’état


Eco-friendly Biomanufacturing of Flavonoid Dyes in Escherichia coli via Computer-mediated Optogenetic Regulation


In this section we describe the problem, our Coup Dy'état project as well as an overview of the synthetic biology.



The Problem

Water pollution is a key problem in the textile dyeing and dyestuff production industry. In many regions of the world, rivers and water bodies that are sources of drinking water and livelihood are becoming heavily polluted by textile dyeing. This is largely due to the irresponsible disposal of industrial effluents, hard-to-biodegrade synthetic dyes by textiles and dyestuff producers and ineffective wastewater treatment.


Image 1. Citarum River. A villager navigates his way through heavily dye-polluted waters. Photo: Iqbal Ksumadireza, The Straits Times, 2018

What is Happening

Already, the Citarum River in Indonesia is a clear example of this murky problem. The 30 million residents relying on the river as their only water source and livelihood are experiencing adverse skin conditions and increased exposure to infectious diseases[1], while the river has almost no aquatic life left[2]. According to a 2015 UN report, in some areas of the river, lead levels at more than 1,000 times the USEPA standard in drinking water have been found[3].


Image 2. "In some areas of the river, lead levels at more than 1,000 times the USEPA standard in drinking water."

River water rapidly changing colours from red, to green, yellow, and black due to high concentrations of dye is not an uncommon sight. However, Citarum River is not an isolated case. Similar sights can be seen in Pearl River in China, Buriganga River in Bangladesh, and Bagmati River in India. In Dhaka, Bangladesh, 719 factories and textile mills generate close to 200 metric tons of wastewater per ton of fabric per year[4]. According to the World Bank, on the global scale, the textile dyeing and dyestuff production industry is the second most pollutive industry, coming in only after oil, and produces a fifth of the world’s water pollution. It also uses the most water apart from agriculture[5].


Image 3. "...textile dyeing and dyestuff production industry is the second most pollutive industry."

Addressing this problem through legislation has not been effective and treatment of the waste is expensive. In recent years, some efforts have been put in to revive natural dyes as a more sustainable alternative to the synthetic dyes[6]. However, this motion has not been gaining much traction, as traditional natural dye production which extracts dye from plants faces many constraints. Natural dyes produced traditionally often have inconsistent quality, varying from batch to batch of plants used. Furthermore, it is land and labour intensive and competes with food production for land use.


Image 4. "...it (natural dye production) is land and labour intensive and competes with food production for land use."



Our Motivation


Our team strongly believes that the current approach of producing synthetic dyes is unsustainable. The world urgently needs a more sustainable and eco-friendly solution. To this end, we have taken the synthetic biology approach to natural dye bioproduction in the hopes of making natural dyes a better substitute for synthetic dyes. What this means is our dyes have to be non-toxic, have reduced use of chemicals in its production, environmentally friendly and still remain appealing to fashion designers and consumers.


Our project entitled Coup Dy'état aims to develop a new bio-manufacturing method of producing flavonoids in E. coli for use as natural dyes.


Image 5. Our dyes be non-toxic, have reduced use of chemicals in its production, environmentally friendly and still remain appealing to fashion designers and consumers

The Challenges


However, in current bio-manufacturing methods, constraints and limitations prevent bio-manufacturing from achieving more of its potential. One limitation is cost, as expensive chemical inducers and feedstock are often required during the bio-manufacturing. Chemical inducers are often used in production to activate gene expression when cells have reached high cell density. The feedstock can make up as much as 18% of total production costs[7].


Although environmental stresses are currently taken into consideration and managed in bioreactors, there is one form of cell stress that is often neglected. Stress induced by the expression of recombinant proteins is present not only in large-scale bioreactor, but also in bench-top laboratory experiments. The depletion of finite cellular resources during the expression of synthetic constructs constitutes an unwanted burden, hampering the growth and expected performance of engineered cells in an unpredictable manner[8]. The bio-manufacturing process can consume as much as 25% of all cellular transcriptional resources[9]. Unfortunately, there is a lack of monitoring for such cell stress within industrial bioreactors, leading to cell death that is counterproductive to bio-manufacturing.




Our Solution


Figure 1. Solution Overview
Top row (from left): Xylose, De Novo Biosynthesis of Naringenin, Luteolin production.
Bottom row (from left):Blue light repressible system, RFP cell stress reporter, cell-machine interface

Figure 2. Platform solution developed

To address the challenges, our team decided to develop a computer-mediated optogenetic regulation system that serves as a platform for future bio-manufacturing and biosynthesis. The system includes an engineered cell in which its biosynthesis pathway can be rapidly regulated by light and having burden-based cellular stress monitored and regulated by a specially designed hardware.


Firstly, to eliminate the use of expensive chemical inducers to switch from growth to the production phase, and we aim to use optogenetic control. To allow dynamic gene regulation in our engineered bacteria, we designed an optogenetic circuit using a blue light repressible promoter for flavonoid biosynthesis. With proper positioning of blue light LEDs, by switching on and off blue light, we can achieve more precise and effective activating and repressing gene activity. Unlike chemical inducers, light can be easily and quickly removed from the bioreactor to allow for de-repression of gene activity.


We also engineered our cells to promote xylose uptake in order to enable the use of lignocellulosic waste as a much more sustainable feedstock for our cells. Lignocellulosic waste is a renewable source of biomass from which xylose can be processed from. The use of xylose over glucose is also advantageous, as metabolism of glucose will result in the production of acetic acid, a growth inhibitor[10]. This could cause bio-manufacturing to be less efficient, achieve lower yield and make it less economically viable.



Secondly, as it is critical to monitor cellular stress induced by the expression of recombinant proteins for efficient production, we introduced a stress-sensing fluorescence reporter. Placed under a burden-responsive promoter, the expression of RFP will increase in proportion with cellular burden, giving us the much-needed feedback in order to take corrective actions to reduce cell stress by decreasing the expression of the synthetic construct.


Lastly, in order to enhance the feedback operation and ensure bio-manufacturing is constantly optimised and at its most optimal state, a computer-aided system was developed to automatically regulate gene expression using light according to the feedback from the stress sensor. This creates a closed-loop system that is devoid of manual inputs. The metabolic burden from the expression of recombinant proteins is dynamic and constantly changing, and the regulation of cell stress can only be made possible through the use of a closed-loop feedback system, which is what we have achieved. To demonstrate this approach, we produced Luteolin, a natural yellow dye.



References


[1] France-Presse, Agence. Indonesia takes on task of cleaning up 'world's dirtiest river'. 2 March, 2018. 3 July, 2018. https://www.thenational.ae/world/asia/indonesia-takes-on-task-of-cleaning-up-world-s-dirtiest-river-1.709520

[2] Asian Development Bank; The World Bank. Downstream Impacts of Water Pollution in the Upper Citarum River, West Java, Indonesia. Technical Paper. Manila: Asian Development Bank, 2013. Document. https://www.adb.org/sites/default/files/publication/154493/citarum-river-downstream-impacts-water-pollution.pdf

[3] Visvanathan, C. "3Rs for Water Security in Asia and the Pacific." United Nations Centre for Regional Development. 2015. 17. Document. http://www.uncrd.or.jp/content/documents/2660Pre-Final-BG-Plenary%20Session-6_FINAL.pdf

[4] Chequer, F. M., de Oliveira, G. A., Ferraz, E. R., Cardoso, J. C., Zanoni, M. V., & de Oliveira, D. P. (2013). Textile Dyes: Dyeing Process and Environmental Impact. IntechOpen, 151-176.

[5] Asian Development Bank; The World Bank. (2013). Downstream Impacts of Water Pollution in the Upper Citarum River, West Java, Indonesia. Manila: Asian Development Bank. Retrieved from https://www.adb.org/sites/default/files/publication/154493/citarum-river-downstream-impacts-water-pollution.pdf

[6] Křížová, Hana. (2015). Natural dyes: their past, present, future and sustainability. 59-71.

[7] Choi, W. (2018, Oct 11). Cost of Feedstock in Bio-based manufacturing. (A. Leow, Interviewer)

[8] Gasser, B., Saloheimo, M., Rinas, U., Dragosits, M., Rodríguez-Carmona, E., Baumann, K., ... & Porro, D. (2008). Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microbial cell factories, 7(1), 11.

[9] Ceroni, F., Boo, A., Furini, S., Gorochowski, T. E., Borkowski, O., Ladak, Y. N., ... & Ellis, T. (2018). Burden-driven feedback control of gene expression. Nature methods, 15(5), 387.

[10] Bäcklund et al. Microbial Cell Factories 2011, 10:35 http://www.microbialcellfactories.com/content/10/1/35



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