Difference between revisions of "Team:Cornell/Collaborations"

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                <a href="https://2017.igem.org/Team:Cornell"><img src="https://static.igem.org/mediawiki/2017/1/1d/CornellOxyponicsLogo.png" alt="Oxyponics"></a>
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              <a href="https://2017.igem.org/Team:Cornell"><img src="https://static.igem.org/mediawiki/2017/1/1d/CornellOxyponicsLogo.png" alt="Oxyponics"></a>
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              <li><a href="https://2017.igem.org/Team:Cornell">HOME</a></li>
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              <li><a href="https://2017.igem.org/Team:Cornell/Description">ABOUT</a></li>
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            <li><a href="https://2017.igem.org/Team:Cornell">HOME</a></li>
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              <li class="mega-menu menu-5-col"><a href="#">TOOLKIT</a>
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            <li><a href="https://2017.igem.org/Team:Cornell/Description">ABOUT</a></li>
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            <li class="mega-menu menu-5-col"><a href="#">TOOLKIT</a>
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                <li class="menu-title"><a href="#">WET LAB</a>
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                      <li><a href="https://2017.igem.org/Team:Cornell/Contribution">CONTRIBUTION</a></li>
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                      <li><a href="https://2017.igem.org/Team:Cornell/Parts">PARTS</a></li>
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                    <li><a href="https://2017.igem.org/Team:Cornell/Demonstrate">DEMONSTRATE</a></li>
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                    <li><a href="https://2017.igem.org/Team:Cornell/Contribution">CONTRIBUTION</a></li>
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                      <li><a href="https://2017.igem.org/Team:Cornell/Protocol">PROTOCOLS</a></li>
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                     <li><a href="https://2017.igem.org/Team:Cornell/Protocol">PROTOCOLS</a></li>
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                  <li class="menu-title"><a href="#">PRODUCT DEVELOPMENT</a>
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                      <li><a href="https://2017.igem.org/Team:Cornell/Design">DESIGN PROCESS</a></li>
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                  <li class="menu-title"><a href="#">DOCUMENTATION</a>
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              <li><a href="#">HUMAN CENTERED DESIGN</a>
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                  <li><a href="https://2017.igem.org/Team:Cornell/HP/Gold_Integrated">PRACTICES</a></li>
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                  <li><a href="https://2017.igem.org/Team:Cornell/Policies">POLICIES</a></li>
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                  <li><a href="https://2017.igem.org/Team:Cornell/Entrepreneurship">ENTREPRENEURSHIP</a></li>
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                  <li><a href="https://2017.igem.org/Team:Cornell/Collaborations">COLLABORATIONS</a></li>
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                    <li><a href="https://2017.igem.org/Team:Cornell/Design">DESIGN PROCESS</a></li>
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                  <li><a href="https://2017.igem.org/Team:Cornell/Engagement">PUBLIC ENGAGEMENT</a></li>
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                    <li><a href="https://2017.igem.org/Team:Cornell/Software">SOFTWARE</a></li>
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                    <li><a href="https://2017.igem.org/Team:Cornell/Model">MODELING</a></li>
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                  <li><a href="https://2017.igem.org/Team:Cornell/Team">BIOS</a></li>
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                  <li><a href="https://2017.igem.org/Team:Cornell/Attributions">ATTRIBUTIONS</a></li>
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            <li><a href="#">HUMAN CENTERED DESIGN</a>
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                <li><a href="https://2017.igem.org/Team:Cornell/HP/Gold_Integrated">PRACTICES</a></li>
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                <text text-anchor="middle" alignment-baseline="middle" text-anchor="middle" x=50% y=50% style="font-family:Crimson Text" font-size="65">Collaborations</text>
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                     <li><a href="#overview">OVERVIEW</a></li>
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                    <li><a href="#biobricks">BIOBRICKS</a></li>
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                    <li><a href="#chassis">CHASSIS</a></li>
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                    <li><a href="#results">RESULTS</a></li>
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                    <li><a href="#futurework">FUTURE WORK</a></li>
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                <li><a href="https://2017.igem.org/Team:Cornell/Collaborations">COLLABORATIONS</a></li>
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            <li><a href="#">TEAM</a>
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                <li><a href="https://2017.igem.org/Team:Cornell/Team">BIOS</a></li>
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                <li><a href="https://2017.igem.org/Team:Cornell/Attributions">ATTRIBUTIONS</a></li>
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                      <div class="content-title"><a id="overview">OVERVIEW</a></div>
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                          <p>Oxyponics was conceived in the summer of 2017 by Cornell iGEM - a synthetic biology engineering project team composed of 36 undergraduate students. The company created a toolkit to optimize redox state by controlling the amount of reactive oxygen species in the environment. Utilizing the research conducted by the team throughout 2017, Oxyponics created a novel biosensor that couples a redox-sensitive fluorescent protein and an optogenetic circuit to a spectrometric receiver. This allows for a dual reporter-response functionality that greatly enhances versatility and sensitivity over traditional biosensor systems. With the development of an effective large-scale sensing system to monitor plants and track data, the optimal level of oxidative stress that can be determined for the plant systems. Members of the team communicated with local farmers to learn more about Through the feedback of farmers themselves, we created Oxyponics, which couples fluorescent bacteria with a camera-rail system for imaging, to provide exactly the large-scale blanket-type sensing that conventional redox probes cannot.
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        <text text-anchor="middle" alignment-baseline="middle" text-anchor="middle" x=50% y=50% style="font-family:Crimson Text" font-size="65">Contributions</text>
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                      <div class="content-title"><a id="biobricks">BIOBRICKS</a></div>
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                        <p>Oxyponics was conceived in the summer of 2017 by Cornell iGEM - a synthetic biology engineering project team composed of 36 undergraduate students. The company created a toolkit to optimize redox state by controlling the amount of reactive oxygen species in the environment. Utilizing the research conducted by the team throughout 2017, Oxyponics created a novel biosensor that couples a redox-sensitive fluorescent protein and an optogenetic circuit to a spectrometric receiver. This allows for a dual reporter-response functionality that greatly enhances versatility and sensitivity over traditional biosensor systems. With the development of an effective large-scale sensing system to monitor plants and track data, the optimal level of oxidative stress that can be determined for the plant systems. Members of the team communicated with local farmers to learn more about Through the feedback of farmers themselves, we created Oxyponics, which couples fluorescent bacteria with a camera-rail system for imaging, to provide exactly the large-scale blanket-type sensing that conventional redox probes cannot.
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                      <div class="content-title"><a id="chassis">CHASSIS</a></div>
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                        <p>Oxyponics was conceived in the summer of 2017 by Cornell iGEM - a synthetic biology engineering project team composed of 36 undergraduate students. The company created a toolkit to optimize redox state by controlling the amount of reactive oxygen species in the environment. Utilizing the research conducted by the team throughout 2017, Oxyponics created a novel biosensor that couples a redox-sensitive fluorescent protein and an optogenetic circuit to a spectrometric receiver. This allows for a dual reporter-response functionality that greatly enhances versatility and sensitivity over traditional biosensor systems. With the development of an effective large-scale sensing system to monitor plants and track data, the optimal level of oxidative stress that can be determined for the plant systems. Members of the team communicated with local farmers to learn more about Through the feedback of farmers themselves, we created Oxyponics, which couples fluorescent bacteria with a camera-rail system for imaging, to provide exactly the large-scale blanket-type sensing that conventional redox probes cannot.
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                      <div class="content-title"><a id="results">RESULTS</a></div>
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                        <p>Oxyponics was conceived in the summer of 2017 by Cornell iGEM - a synthetic biology engineering project team composed of 36 undergraduate students. The company created a toolkit to optimize redox state by controlling the amount of reactive oxygen species in the environment. Utilizing the research conducted by the team throughout 2017, Oxyponics created a novel biosensor that couples a redox-sensitive fluorescent protein and an optogenetic circuit to a spectrometric receiver. This allows for a dual reporter-response functionality that greatly enhances versatility and sensitivity over traditional biosensor systems. With the development of an effective large-scale sensing system to monitor plants and track data, the optimal level of oxidative stress that can be determined for the plant systems. Members of the team communicated with local farmers to learn more about Through the feedback of farmers themselves, we created Oxyponics, which couples fluorescent bacteria with a camera-rail system for imaging, to provide exactly the large-scale blanket-type sensing that conventional redox probes cannot.
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                        </p>
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                      <div class="content-title"><a id="futurework">FUTURE WORKS</a></div>
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                        <p>Oxyponics was conceived in the summer of 2017 by Cornell iGEM -  a synthetic biology engineering project team composed of 36 undergraduate students. The company created a toolkit to optimize redox state by controlling the amount of reactive oxygen species in the environment. Utilizing the research conducted by the team throughout 2017, Oxyponics created a novel biosensor that couples a redox-sensitive fluorescent protein and an optogenetic circuit to a spectrometric receiver. This allows for a dual reporter-response functionality that greatly enhances versatility and sensitivity over traditional biosensor systems. With the development of an effective large-scale sensing system to monitor plants and track data, the optimal level of oxidative stress that can be determined for the plant systems. Members of the team communicated with local farmers to learn more about Through the feedback of farmers themselves, we created Oxyponics, which couples fluorescent bacteria with a camera-rail system for imaging, to provide exactly the large-scale blanket-type sensing that conventional redox probes cannot.
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                      <div class="content-title"><a id="references">REFERENCES</a></div>
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                        <p>Oxyponics was conceived in the summer of 2017 by Cornell iGEM - a synthetic biology engineering project team composed of 36 undergraduate students. The company created a toolkit to optimize redox state by controlling the amount of reactive oxygen species in the environment. Utilizing the research conducted by the team throughout 2017, Oxyponics created a novel biosensor that couples a redox-sensitive fluorescent protein and an optogenetic circuit to a spectrometric receiver. This allows for a dual reporter-response functionality that greatly enhances versatility and sensitivity over traditional biosensor systems. With the development of an effective large-scale sensing system to monitor plants and track data, the optimal level of oxidative stress that can be determined for the plant systems. Members of the team communicated with local farmers to learn more about Through the feedback of farmers themselves, we created Oxyponics, which couples fluorescent bacteria with a camera-rail system for imaging, to provide exactly the large-scale blanket-type sensing that conventional redox probes cannot.
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                    <li><a href="#part1">Part: BB<span class="lowercase">a</span>_K1075044</a></li>
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                    <li><a href="#part2">Part: BB<span class="lowercase">a</span>_K1758335</a></li>
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                   <div class="content-title top"><a id="part1">Part: BB<span class="lowercase">a</span>_K1075044</a></div>
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                  <p>In implementing an inducible expression system, we chose to incorporate pDawn and pDusk as optogenetic promoters to affect the expression of superoxide dismutase.
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                  <p>We can characterize pDawn and pDusk by Western blot. By growing E. coli under different intensities of visible light, we can compare the relative expression levels of a FLAG-tagged superoxide dismutase under optogenetic control.
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                  <p>We first inserted superoxide dismutase into the pDusk circuit. We then proceeded to incorporate a cI repressor (BBa_K2296045) and a pR promoter into the pDusk system followed by insertion of superoxide dismutase to create a light-inducible pDawn circuit.
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                  <p>We assessed the transcriptional activity of pDawn and pDusk in response to three different levels of light intensity from a standard laboratory fluorescent light:
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                    <img src="https://static.igem.org/mediawiki/2017/3/31/T--Cornell--WetLab_DoseResponse.png" alt="Dose Response"/>
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                  <p>Our initial characterization revealed a dose-dependence of pDusk. However, pDawn did not display a dose-dependent response in response to stimulation from fluorescent light. We suspect this to be the result of low light intensity.
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              <!------------------------END FOOTER------------------------>
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                  </p>
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      </main>
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                  <p>Our subsequent characterization uses a significantly greater intensity of LED light:</p>
 +
                  <div class="image-wrapper center">
 +
                    <img src="https://static.igem.org/mediawiki/2017/5/59/T--Cornell--WetLab_pDawnpDuskExpression.png" alt="pDawnpDuskExpression"/>
 +
                  </div>
 +
                  <p>Our results suggest a light-intensity dependence of pDusk and pDawn. Our results also show that our cI repressor biobrick successfully converted the light-repressible pDusk into a light-inducible pDawn.
 +
                  </p>
 +
                  <p>In addition to dose-dependent characterization, we also conducted time-dependence characterization of pDawn following stimulation by 470 nm LED light (LTL3H3TBPADS1) with the following emission spectrum [1]:</p>
 +
                  <div class="image-wrapper center">
 +
                    <img src="https://static.igem.org/mediawiki/2017/6/6e/470LEDAbsSpec.png" alt="470LEDAbsSpec"/>
 +
                  </div>
 +
                  <p>Our initial characterization was conducted with 1 second pulses and revealed a significant lag between initial exposure and translational response:</p>
 +
                  <div class="image-wrapper">
 +
                    <img class="img-responsive center" src="https://static.igem.org/mediawiki/2017/a/a3/T--Cornell--WetLab_pDawnTimeDependence.png" alt="pDawnTimeDependence"/>
 +
                  </div>
 +
                  <p>For our subsequent characterization, we increased the intensity and pulse duration of the LED to parse out more subtle effects at earlier time points:
 +
                  </p>
 +
                  <div class="image-wrapper center">
 +
                    <img src="https://static.igem.org/mediawiki/2017/0/03/T--Cornell--WetLab_pDawnTimeDependenceGraph.png" alt="pDawnTimeDependence"/>
 +
                  </div>
 +
                  <p>Our results suggest a light intensity and time-dependence of the pDawn and pDusk optogenetic circuit. </p>
 +
                  <div class="content-title"><a id="part2">Part: BB<span class="lowercase">a</span>_K1758335</a></div>
 +
                  <p>We initially pursued this part as part of a of detector for toxins by cigarette smoke.  Upon failure to observe an appreciable difference in expression as well as a transition to a different overarching application, we abandoned this aspect of the project and no further testing was conducted.
 +
                  </p>
 +
                  <p>Characterization was conducted on this part to determine the plausibility of using it for detection of low concentrations of lead.  To this effect, we characterized expression at three different concentrations of lead and documented this on a gel as seen below.  The concentrations are as follows:  0, 0.012, 0.024, 0.072 glL Pb(NO3)2.
 +
                  </p>
 +
                  <div class="image-wrapper">
 +
                    <img class="img-responsive center" src="https://static.igem.org/mediawiki/2017/c/cf/T--Cornell--WetLab_mRFPCharacterization.png" alt="mRFPCharacterization" width="50%"/>
 +
                  </div>
 +
                  <p>As can be seen from the gel, no appreciable difference was observed in any of the samples.  While we did not conduct further testing, preliminary data suggests that this part does not function as described.
 +
                  </p>
 +
                  <p>Furthermore, it should be noted that the construct contains mRFP and not sfGFP as the name might suggest, which was confirmed by BLAST.
 +
                  </p>
 +
 
 +
                  <div class="content-title"><a id="references">References</a></div>
 +
                  <ol class="references">
 +
                    <li>Lite-On Technology Corporation. Information Data Sheet for LED Lamp LTL3H3TBPADS1-132A. Retrieved from http://www.mouser.com/ds/2/239/Lite-On_LTL3H3TBPADS1-132A-Ver.A-341105.pdf </li>
 +
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Revision as of 02:04, 29 October 2017

<!DOCTYPE html> Contribution

Contributions
Part: BBa_K1075044

In implementing an inducible expression system, we chose to incorporate pDawn and pDusk as optogenetic promoters to affect the expression of superoxide dismutase.

We can characterize pDawn and pDusk by Western blot. By growing E. coli under different intensities of visible light, we can compare the relative expression levels of a FLAG-tagged superoxide dismutase under optogenetic control.

We first inserted superoxide dismutase into the pDusk circuit. We then proceeded to incorporate a cI repressor (BBa_K2296045) and a pR promoter into the pDusk system followed by insertion of superoxide dismutase to create a light-inducible pDawn circuit.

We assessed the transcriptional activity of pDawn and pDusk in response to three different levels of light intensity from a standard laboratory fluorescent light:

Dose Response

Our initial characterization revealed a dose-dependence of pDusk. However, pDawn did not display a dose-dependent response in response to stimulation from fluorescent light. We suspect this to be the result of low light intensity.

Our subsequent characterization uses a significantly greater intensity of LED light:

pDawnpDuskExpression

Our results suggest a light-intensity dependence of pDusk and pDawn. Our results also show that our cI repressor biobrick successfully converted the light-repressible pDusk into a light-inducible pDawn.

In addition to dose-dependent characterization, we also conducted time-dependence characterization of pDawn following stimulation by 470 nm LED light (LTL3H3TBPADS1) with the following emission spectrum [1]:

470LEDAbsSpec

Our initial characterization was conducted with 1 second pulses and revealed a significant lag between initial exposure and translational response:

pDawnTimeDependence

For our subsequent characterization, we increased the intensity and pulse duration of the LED to parse out more subtle effects at earlier time points:

pDawnTimeDependence

Our results suggest a light intensity and time-dependence of the pDawn and pDusk optogenetic circuit.

Part: BBa_K1758335

We initially pursued this part as part of a of detector for toxins by cigarette smoke. Upon failure to observe an appreciable difference in expression as well as a transition to a different overarching application, we abandoned this aspect of the project and no further testing was conducted.

Characterization was conducted on this part to determine the plausibility of using it for detection of low concentrations of lead. To this effect, we characterized expression at three different concentrations of lead and documented this on a gel as seen below. The concentrations are as follows: 0, 0.012, 0.024, 0.072 glL Pb(NO3)2.

mRFPCharacterization

As can be seen from the gel, no appreciable difference was observed in any of the samples. While we did not conduct further testing, preliminary data suggests that this part does not function as described.

Furthermore, it should be noted that the construct contains mRFP and not sfGFP as the name might suggest, which was confirmed by BLAST.

References
  1. Lite-On Technology Corporation. Information Data Sheet for LED Lamp LTL3H3TBPADS1-132A. Retrieved from http://www.mouser.com/ds/2/239/Lite-On_LTL3H3TBPADS1-132A-Ver.A-341105.pdf