Difference between revisions of "Team:UC Davis/Design"

 
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      <div style = 'padding-left: 100PX; padding-bottom: 30px; font-size: 40px; color: #d9a900'; ><b>Project Design</b></div>
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      <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>I. Superfund Program</b></div>
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        The United States Environmental Protection Agency (EPA) is the government agency responsible for protection of the natural environment and human health [1]. In 1980, Congress formed the Comprehensive Environmental Response, Compensation
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        and Liability Act (CERCLA), more commonly referred to as the Superfund program. The Superfund program identified the most hazardous, contaminated sites in the United States, and marked them as priorities for clean-up, giving the EPA the authority
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        to step in. There are more than 1300 Superfund sites across the country, and have been linked to increased rates of cancer and other dangers to human health [2]. Currently, nearly one in six Americans live within three miles of a superfund site
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        [3].
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        <br><br>At the University of California, Davis, there is an established group of researchers who have been working with the EPA for the past 31 years to “acquire a better understanding of the human and ecological risks of hazardous substances;
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        and advance the development of new technologies for the cleanup of contaminated sites” [4]. We had the opportunity to join UC Davis researchers on a trip to visit a Native American tribe in northern California who live on heavily polluted land.
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          Visit with Native American Tribe
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          The tribe has reported unusually elevated rates of cancer and miscarriage incidence, and has reason to suspect that the cause may be tied to environmental pollution on their tribal land from local agricultural and forestry corporations. Researchers from UC Davis have been collaborating with the tribe’s scientists and governing council to gather data pertaining to environmental and human health.
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        <br><br>
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        In the United States, recognized Native American tribes are self-governing bodies, and have the power to make and enforce laws and regulations on their own lands. The specific Native American tribe which we visited has expressed their position on genetic engineering in an ordinance adopted in 2015, which may be accessed here [5]. In the ordinance, the tribe makes clear that they view the release of genetically modified organisms into their environment to be a major threat to their cultural values and traditional way of life. Compared to the United States as a whole, which has relatively tolerant laws regarding the production and use of genetically modified organisms, the tribe has far stricter laws.
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        Interestingly, within the ordinance, the tribe makes several exceptions. The first is unusual: “Genetically engineered or modified organisms do not include organisms created by traditional selective breeding, [...] or microorganisms created by moving genes or gene segments between unrelated bacteria” [5].  As much of biotechnology and synthetic biology uses bacteria as a host, we were surprised to find that the ordinance deemed the majority of the work done in the iGEM competition as acceptable.
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        The ordinance also provides exceptions to the prohibition for “State or federally licensed medical research institutions, medical laboratories, or medical manufacturing facilities engaged in licensed medical production, or medical research involving genetically engineered or genetically modified organisms,” as well as for, “Educational or scientific institutes” [5]. This makes it appear that the major focus of the ordinance is to restrict commercial biotechnology and agriculture firms and their crops/livestock on tribal lands. The ordinance specifically refers to transgenic salmon-- which are referred to as a threat to their way of life-- and the significance of the wild salmon to the tribe’s cultural values.
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        After consideration, we came to the the conclusion that a device or solution made by an iGEM team with the purpose of being introduced into the environment would be met with strong resistance, and would be unlikely to benefit society if it were never allowed to be used. Also, we considered the stance of the tribe, concerning the introduction of genetically modified organisms as a threat to their cultural values and traditional way of life. While many arguments made by opponents of GMOs focus on perceived threats to human health, which can be settled empirically by careful in vivo studies, cultural arguments cannot be dismissed as easily. A community should have the right to live according their values and uphold traditional ways of life. If certain communities decide that their values are incompatible with the introduction of genetically modified organisms on to their land, then their decision should be respected.
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        The visit with the Native American tribe helped us focus our project and become aware of human and environmental health problems for which synthetic biology can provide tools to help resolve. While working on the campus of our university, we were subject only to federal (America), state (California), and local (Yolo County, City of Davis, University of California) laws. If we were to return to test our device on tribal lands, we would be required to follow their specific ordinances and regulations, including seeking prior written permission to use genetically engineered devices for biomedical research. Likewise, it would be necessary to seek prior written permission before testing environmental samples taken from tribal lands. A similar procedure would be required when working with other communities.
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        With our project thus contained within the lab, we considered how best to use synthetic biology to help the tribe and other communities facing similar problems with environmental pollution. The agricultural and forestry corporations in the region surrounding the tribe’s land are currently operating within legal regulations, however the tribe has indicated that these regulations are not as strict as they would like. One example a tribal member provided was that currently, herbicides may be applied within fifty feet of sources of drinking water. A concern is that this distance is not sufficient to prevent contamination of drinking water supplies. A variety of harmful chemicals have been found in the waters of the tribal lands, particularly microcystin toxins and organochlorine pesticides [6]. Analysis of water samples by the Young Lab at UC Davis in 2017 also found the presence of low concentrations of pharmaceuticals, including warfarin, in the waters of the tribal lands.
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        If the working hypothesis is found to be supported, that the tribe’s health crises are linked to environmental pollution of their lands and water, then the remedy would be to tighten regulations concerning the use of pesticides, herbicides, and other potentially harmful compounds. If this working hypothesis is not supported by further study, alternative explanations for the tribe’s health crises should be explored, including predisposing genetic factors within the population and other factors.
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        To help collect data to support the working hypothesis, and similar projects involving public health and environmental toxicology, we decided to develop a way to easily test what effect low concentrations of potentially harmful chemicals have on the physiological health of mammalian cells.  
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    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>II. Bioassay Design</b></div>
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        We designed a mammalian cell-based bioassay that reports activation of specific stress pathways via fluorescence, for use in environmental toxicology. To do this, we selected transcriptionally regulated target genes which are present in mammalian
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        cells and are involved in stress pathways. We isolated the promoters with transcription factor binding sites from these target genes and coupled them to a fluorescent reporter gene. We selected EGFP, a variant of green fluorescent protein (GFP).
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        GFP is ubiquitous in synthetic biology due to its reliability and ease of measurement [7]. EGFP is derived from GFP, and has been optimized for use in mammalian systems. When a chemical of concern is screened using our assay, it will trigger a
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        specific stress response, and the reporter gene will be expressed, causing the assay to fluoresce. The fluorescence of the assay can be quantitatively measured and analyzed. This assay will provide data on the effect of chemicals of concern on
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        the physiological health of mammalian cells; measurements may be easily taken a range of concentrations, durations of exposure, salinities, pH, temperatures, nutrient availabilities, and other conditions. This also allows for measurement of synergistic
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        or interfering effects due to multiple chemicals of concern present simultaneously.
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        <br><br> We selected 9 promoter constructs derived from 6 target genes (see Figure 2 below) and coupled them to EGFP. This promoter and reporter gene construct was inserted into a plasmid and transfected into two cell lines (see Figure 3 below).
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        The resulting 18 bioassays were exposed to 6 different chemicals of concern at a variety of concentrations and conditions (see Figure 4 below).
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          Why not use whole organisms?
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      A cell-based approach cannot replace in vivo toxicology studies. However these studies require extensive funding, time, and other resources. By developing a relatively low-cost, cell-based bioassay, preliminary data may be quickly gathered,
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        allowing for more informed decision making as to which in vivo studies are necessary. By using a cell-based preliminary assay, it is our hope that researchers will be able to quickly gather data, make more informed decisions, and save resources.
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        Our cell-based bioassay may also be used to add to the body of knowledge concerning the effect of specific chemicals of concern on the physiological health of mammalian cells and the mechanism of stress.
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            Why not use cell-free biochemical assays, such as ELISA?
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      Antibody-based assays, such as Enzyme-Linked Immunosorbent Assay (ELISA), have been very successful in biomedical research, environmental toxicology, and other fields [8]. These cell-free methods involve selective binding of a target molecule to a prepared antibody. Such methods are very successful at identifying single, known compounds in an environmental sample, but do not provide any data regarding the effect of the chemical of concern on the health of a living cell. Similarly, the tools of analytical chemistry and organic chemistry may be used to great success when identifying molecules, but do not provide any data regarding the actual effect on a living cell.
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          Why use synthetic biology?
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        The goal of our bioassay is to create a tool that can be used to better understand the effect on the physiological health of mammalian cells of environmental toxins. An alternative way to achieve this knowledge is to expose cells to the
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        chemicals of concern, lyse the cells, isolate the RNA, and run a quantitative-real-time-reverse-transcript-PCR in order to characterize and quantify the mRNAs present in the cell. However, this approach has limitations. It necessarily involves
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        lysing the cells, and cannot be used to gather real-time data about the behavior of the same cell over time. By using a fluorescent reporter gene, we can measure the induction of the reporter gene over time without lysing cells, and can more easily
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        take a large number of data points across different chemicals of concern, concentrations, and other variables. A fluorescent bioassay also reduces the amount of work required to measure many data points, compared to PCR based methods.
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          Why use mammalian cells?
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        <a href="#1">Bioassay Design</a>
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        <a href="#2">Promoter Constructs</a>
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        <a href="#3">Host Strains</a>
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        <a href="#4">Chemicals of Concern</a>
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        <a href="#5">Plasmid</a>
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        <a href="#6">Measurement</a>
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        <a href="#7">Extensions</a>
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        <a href="#8">Project Design Notebook</a>
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        <a href="#9">References</a>
  
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      <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' > We chose to use mammalian cells because they make much more accurate models for human health than bacteria or yeast. Furthermore, within the iGEM competition and the field of synthetic biology as a whole, there has been relatively little
 
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        work with mammalian systems, compared to bacteria, yeast, and algae. Working with mammalian cells brings a variety of new challenges and opportunities to iGEM: they are more difficult and expensive to culture than bacteria, they require specialized
 
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        equipment and safety training, they can be used to produce proteins suitable for use in human therapeutics (due to similar most-translational modifications), they can be used for more complicated circuits and pathways utilizing spatial/temporal
 
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        differentiation, and they are much more sensitive to chemicals in the environment (allowing for more sensitive biosensors and bioassays).
 
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          Why not use human cells?
 
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      Although human cell lines would make a superior model for human disease, compared to cell lines derived from hamsters and mice, for our project we chose not to use human cells. Work involving human cells requires specialized facilities,
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        equipment, resources, and safety training. Human cells require a BSL-2 lab, which would have been more difficult for our team to use than our regular wetlab space, which is BSL-1. Additionally, as our project took place in the context of the iGEM
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        competition, we wanted for other teams to be able to easily reproduce our findings and expand them. By using human cell lines, many teams which lack access to a BSL-2 lab, would have had more difficulty in expanding upon our project. It would
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        be relatively straightforward to insert our genetic constructs to a human cell line, and this presents an opportunity to extend our project.
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     <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>III. Promoter Constructs</b></div>
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          Figure 2 shows the promoter constructs we used and the target genes from which they were derived. Full FASTA sequences for our promoter constructs are available here .
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      We designed a mammalian cell-based bioassay that reports activation of specific stress pathways via fluorescence, for use in environmental toxicology. To do this, we selected transcriptionally regulated target genes which are present in mammalian cells and are involved in stress pathways. We isolated the promoters with transcription factor binding sites from these target genes and coupled them to a fluorescent reporter gene. We selected EGFP, a variant of green fluorescent protein (GFP). GFP is ubiquitous in synthetic biology due to its reliability and ease of measurement [1]. EGFP is derived from GFP and has been optimized for use in mammalian systems. When a chemical of concern is screened using our assay, if it triggers a specific stress response, the reporter gene will be expressed, causing the assay to fluoresce. The fluorescence of the assay can be quantitatively measured and analyzed. This assay will provide data on the effect of chemicals of concern on the physiological health of mammalian cells; measurements may be easily taken a range of concentrations, duration of exposure, salinities, pH, temperatures, nutrient availabilities, and other conditions. This also allows for measurement of synergistic or interfering effects due to multiple chemicals of concern present simultaneously.
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    Figure 2. Promoter Constructs
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<p>We selected 8 promoter constructs derived from 5 target genes (see Figure 1 below) and coupled them to EGFP. This promoter and reporter gene construct were inserted into a plasmid and transfected into two cell lines (see Figure 2 below). The resulting bioassays were exposed to different chemicals of concern at a variety of concentrations and conditions (see Figure 3 below).
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          <th class="tg-9hbo">Construct</th>
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          <td class="tg-yw4l">MT1</td>
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          <td class="tg-yw4l">Metallothionein 1</td>
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          <td class="tg-yw4l">[9]</td>
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          <td class="tg-yw4l">59</td>
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+
−
        </tr>
+
−
        <tr>
+
−
          <td class="tg-yw4l">CYP</td>
+
−
          <td class="tg-yw4l">CYP1A1</td>
+
−
          <td class="tg-yw4l">Mus musculus</td>
+
−
          <td class="tg-yw4l">//</td>
+
−
          <td class="tg-yw4l">[11]</td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td class="tg-yw4l">FGF</td>
+
−
          <td class="tg-yw4l">FGF21</td>
+
−
          <td class="tg-yw4l">Homo sapiens</td>
+
−
          <td class="tg-yw4l">//</td>
+
−
          <td class="tg-yw4l">[12]</td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td class="tg-yw4l">GD153</td>
+
−
          <td class="tg-yw4l">GADD153</td>
+
−
          <td class="tg-yw4l">Cricetulus griseus</td>
+
−
          <td class="tg-yw4l">//</td>
+
−
          <td class="tg-yw4l">[13], [14]</td>
+
−
        </tr>
+
−
        <tr>
+
−
          <td class="tg-yw4l">GD45</td>
+
−
          <td class="tg-yw4l">GADD45α</td>
+
−
          <td class="tg-yw4l">Homo sapiens</td>
+
−
          <td class="tg-yw4l">//</td>
+
−
          <td class="tg-yw4l">[15]</td>
+
−
        </tr>
+
−
      </table>
+
−
    </center>
+
  
 +
<div style='padding-left:10%;padding-right: 7%'>A cell-based approach cannot replace in vivo toxicology studies. However, these studies require extensive funding, time, and other resources. By developing a relatively low-cost, cell-based bioassay, preliminary data may be quickly gathered, allowing for more informed decision making as to which in vivo studies are necessary. By using a cell-based preliminary assay, it is our hope that researchers will be able to quickly gather data, make more informed decisions, and save resources. Our cell-based bioassay may also be used to add to the body of knowledge concerning the effect of specific chemicals of concern on the physiological health of mammalian cells and the mechanism of stress. </div>
 +
<div style='padding-top: 10px;'></div>
 +
<div style = 'padding-left:10%;padding-bottom: 22px; font-size: 20px; color: #d9a900'; ><b><i>Why not use cell-free biochemical assays, such as ELISA?</i></b></div>
  
 +
<div style='padding-left:10%;padding-right: 7%'>
 +
  Antibody-based assays, such as Enzyme-Linked Immunosorbent Assay (ELISA), have been very successful in biomedical research, environmental toxicology, and other fields [2]. These cell-free methods involve selective binding of a target molecule to a prepared antibody. Such methods are very successful at identifying single, known compounds in an environmental sample, but do not provide any data regarding the effect of the chemical of concern on the health of a living cell. Similarly, the tools of analytical chemistry and organic chemistry may be used to great success when identifying molecules, but do not provide any data regarding the actual effect on a living cell.
 +
</div>
 +
<div style='padding-top: 10px;'></div>
 +
<div style = 'padding-left:10%;padding-bottom: 22px; font-size: 20px; color: #d9a900'; ><b><i>Why use synthetic biology?</i></b></div>
 +
<div style='padding-left:10%;padding-right: 7%'>
  
−
    <div style='padding-top: 30px;'></div>
+
The goal of our bioassay is to create a tool that can be used to better understand the effect on the physiological health of mammalian cells of environmental toxins. An alternative way to achieve this knowledge is to expose cells to the chemicals of concern, lyse the cells, isolate the RNA, and run a quantitative-real-time-reverse-transcription-PCR in order to characterize and quantify the mRNAs present in the cell. However, this approach has limitations. It requires lysing the cells and cannot be used to gather real-time data about the behavior of the same cell over time. By using a fluorescent reporter gene, we can measure the induction of the reporter gene over time without lysing cells and can more easily take a large number of data points across different chemicals of concern, concentrations, and other variables. A fluorescent bioassay also reduces the amount of work required to measure many data points, compared to PCR based methods. Our bioassay also makes possible the future study of the behavior of a single cell over time after exposure to a chemical of concern, with the aid of microfluidics.
−
    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>IV. Host Strains</b></div>
+
</div>
  
−
<center>
+
<div style = 'padding-left:10%;padding-bottom: 22px; font-size: 20px; color: #d9a900'; ><b><i>Why use mammalian cells?</i></b></div>
−
  <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' >
+
<div style='padding-left:10%;padding-right: 7%'>
−
    Figure 3: Host Strains
+
  We chose to use mammalian cells because they make much more accurate models for human health than bacteria or yeast. Furthermore, within the iGEM competition and the field of synthetic biology as a whole, there has been relatively little work with mammalian systems, compared to bacteria, yeast, and algae. Working with mammalian cells brings a variety of new challenges and opportunities to iGEM: they are more difficult and expensive to culture than bacteria, they require specialized equipment and safety training, they can be used to produce proteins suitable for use in human therapeutics (due to similar post-translational modifications), and they can be used for more complicated circuits and pathways utilizing spatial/temporal differentiation [3].
  
 +
</div>
 +
<div style='padding-top: 10px;'></div>
 +
<div style = 'padding-left:10%;padding-bottom: 22px; font-size: 20px; color: #d9a900'; ><b><i>Why not use human cells?</i></b></div>
 +
<div style='padding-left:10%;padding-right: 7%'>
 +
  Although human cell lines would make a superior model for human disease, compared to cell lines derived from hamsters and mice, for our project we chose not to use human cells. Work involving human cells requires specialized facilities, equipment, resources, and safety training. Human cells require a BSL-2 lab, which would have been more difficult for our team to use than our regular wetlab space, which is BSL-1. Additionally, as our project took place in the context of the iGEM competition, we wanted for other teams to be able to easily reproduce our findings and expand them. By using human cell lines, many teams, which lack access to a BSL-2 lab, would have had more difficulty in expanding upon our project. It would be relatively straightforward to insert our genetic constructs to a human cell line, and this presents an opportunity to extend our project.
 +
</div>
 +
<div style='padding-top: 20px;'></div>
 +
<div id="2" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Promoter Constructs</b></div>
 +
<div style='padding-top: 10px;'></div>
 +
<div style='padding-left:7%;padding-right: 7%'>
 +
 +
Table 1 shows the promoter constructs we used and the target genes from which they were derived. Full FASTA sequences for our promoters are available <a href= "https://2018.igem.org/Team:UC_Davis/SupplementalMaterials">here.</a>
 +
</div>
 +
<div style='padding-top: 10px;'></div>
 +
<center>
 +
  <div style = 'line-height: 25px' >
 +
    Table 1. Promoter Constructs
 +
<p></p>
 
   </div>
 
   </div>
−
    <style type="text/css">
+
  <style type="text/css">
 
.tg  {border-collapse:collapse;border-spacing:0;border-color:#aaa;}
 
.tg  {border-collapse:collapse;border-spacing:0;border-color:#aaa;}
−
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+
.tg td{font-family:Arial, sans-serif;font-size:12px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#aaa;color:#333;background-color:#fff;}
−
.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#aaa;color:#fff;background-color:#002855;}
+
.tg th{font-family:Arial, sans-serif;font-size:12px;font-weight:normal;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#aaa;color:#fff;background-color:#002855;}
 
.tg .tg-9hbo{font-weight:bold;vertical-align:top}
 
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.tg .tg-yw4l{vertical-align:top}
Line 243: Line 159:
 
<table class="tg">
 
<table class="tg">
 
<tr>
 
<tr>
−
   <th class="tg-9hbo">Host Strain</th>
+
   <th class="tg-9hbo">Construct</th>
−
   <th class="tg-9hbo">Description</th>
+
   <th class="tg-9hbo">Target Gene</th>
−
   <th class="tg-9hbo">Supplier</th>
+
   <th class="tg-9hbo">Species of Origin</th>
 +
  <th class="tg-9hbo">Stress Pathway</th>
 +
  <th class="tg-9hbo">Size</th>
 +
  <th class="tg-9hbo">Further Reading</th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
   <td class="tg-yw4l">CHO-DG44</td>
+
   <td class="tg-yw4l">MT1</td>
−
   <td class="tg-yw4l">An immortal, adherent cell line derived from chinese hamster (Cricetulus griseus) ovary cells. The strain we used is dihydrofolate reductase deficient.</td>
+
  <td class="tg-yw4l">Metallothionein 1</td>
−
   <td class="tg-yw4l">ATCC: CRL-9096 [16]</td>
+
   <td class="tg-yw4l">Mus musculus</td>
 +
  <td class="tg-yw4l">Oxidative, heavy metal</td>
 +
   <td class="tg-yw4l">305 nucleotides</td>
 +
  <td class="tg-yw4l">[4]</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
   <td class="tg-yw4l">AML-12</td>
+
   <td class="tg-yw4l">MT2_1</td>
−
   <td class="tg-yw4l">An immortal, adherent cell line derived from mouse (Mus musculus) liver cells.</td>
+
  <td class="tg-yw4l">Metallothionein 2</td>
−
   <td class="tg-yw4l">ATCC: CRL-2254 [17]</td>
+
   <td class="tg-yw4l">Homo sapiens</td>
 +
  <td class="tg-yw4l">Oxidative, heavy metal</td>
 +
   <td class="tg-yw4l">377 nucleotides</td>
 +
  <td class="tg-yw4l">[5]</td>
 
</tr>
 
</tr>
−
</table>
 
−
</center>
 
−
 
−
 
−
 
−
 
−
    <div style='padding-top: 30px;'></div>
 
−
    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>V.Chemicals of concern </b></div>
 
−
<center>
 
−
  <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' >
 
−
    Figure 4: Chemicals of Concern
 
−
  </div>
 
−
    <style type="text/css">
 
−
.tg  {border-collapse:collapse;border-spacing:0;border-color:#aaa;}
 
−
.tg td{font-family:Arial, sans-serif;font-size:14px;padding:20px 20px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#aaa;color:#333;background-color:#fff;}
 
−
.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:20px 20px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#aaa;color:#fff;background-color:#002855;}
 
−
.tg .tg-9hbo{font-weight:bold;vertical-align:top}
 
−
.tg .tg-yw4l{vertical-align:top}
 
−
</style>
 
−
<table class="tg">
 
 
<tr>
 
<tr>
−
   <th class="tg-9hbo">Chemical of Concern</th>
+
   <td class="tg-yw4l">MT2_2</td>
−
   <th class="tg-9hbo">Class</th>
+
   <td class="tg-yw4l">Metallothionein 2</td>
 +
  <td class="tg-yw4l">Homo sapiens</td>
 +
  <td class="tg-yw4l">Oxidative, heavy metal</td>
 +
  <td class="tg-yw4l">59 nucleotides</td>
 +
  <td class="tg-yw4l">[5]</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
   <td class="tg-yw4l">Copper Sulfate</td>
+
   <td class="tg-yw4l">MT2_3</td>
−
   <td class="tg-yw4l">Heavy metal</td>
+
   <td class="tg-yw4l">Metallothionein 2</td>
 +
  <td class="tg-yw4l">Homo sapiens</td>
 +
  <td class="tg-yw4l">Oxidative, heavy metal</td>
 +
  <td class="tg-yw4l">60 nucleotides; construct MT2_2 plus one base at 5’ end</td>
 +
  <td class="tg-yw4l">[5]</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
   <td class="tg-yw4l">Zinc Sulfate</td>
+
   <td class="tg-yw4l">MT2_4</td>
−
   <td class="tg-yw4l">Heavy metal</td>
+
   <td class="tg-yw4l">Metallothionein 2</td>
 +
  <td class="tg-yw4l">Homo sapiens</td>
 +
  <td class="tg-yw4l">Oxidative, heavy metal</td>
 +
  <td class="tg-yw4l">60 nucleotides; construct MT2_2 plus one base at 3’ end</td>
 +
  <td class="tg-yw4l">[5]</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
   <td class="tg-yw4l">Metam Sodium</td>
+
   <td class="tg-yw4l">FGF</td>
−
   <td class="tg-yw4l">Organosulfur biocide</td>
+
   <td class="tg-yw4l">FGF21</td>
 +
  <td class="tg-yw4l">Homo sapiens</td>
 +
  <td class="tg-yw4l">Unfolded protein response (UPR), endoplasmic reticulum stress</td>
 +
  <td class="tg-yw4l">695 nucleotides</td>
 +
  <td class="tg-yw4l">[6]</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
   <td class="tg-yw4l">2,4-D</td>
+
   <td class="tg-yw4l">GD153</td>
−
   <td class="tg-yw4l">Organochlorine herbicide</td>
+
  <td class="tg-yw4l">GADD153</td>
 +
  <td class="tg-yw4l">Cricetulus griseus</td>
 +
   <td class="tg-yw4l">Organochlorine biocides, genotoxins</td>
 +
  <td class="tg-yw4l">811 nucleotides</td>
 +
  <td class="tg-yw4l">[7], [8]</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
−
   <td class="tg-yw4l">Warfarin</td>
+
   <td class="tg-yw4l">GD45</td>
−
   <td class="tg-yw4l">Pharmaceutical anticoagulant, pesticide</td>
+
   <td class="tg-yw4l">GADD45α</td>
−
</tr>
+
  <td class="tg-yw4l">Homo sapiens</td>
−
<tr>
+
  <td class="tg-yw4l">Genotoxins, mechanical stress</td>
−
   <td class="tg-yw4l">Hydrogen Peroxide</td>
+
   <td class="tg-yw4l">1006 nucleotides</td>
−
   <td class="tg-yw4l">Oxidizing agent</td>
+
   <td class="tg-yw4l">[9]</td>
 
</tr>
 
</tr>
 +
</table>
 +
    </center>
 +
<div style='padding-top: 20px;'></div>
 +
    <div id="3"  style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Host Strains</b></div>
 +
    <div style='padding-top: 10px;'></div>
 +
 +
    <center>
 +
      <div style = 'line-height: 25px' >
 +
        Table 2. Host Strains
 +
    <p></p>
 +
      </div>
 +
 +
 +
      <style type="text/css">
 +
    .tg  {border-collapse:collapse;border-spacing:0;border-color:#aaa;}
 +
    .tg td{font-family:Arial, sans-serif;font-size:12px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#aaa;color:#333;background-color:#fff;}
 +
    .tg th{font-family:Arial, sans-serif;font-size:12px;font-weight:normal;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#aaa;color:#fff;background-color:#002855;}
 +
    .tg .tg-9hbo{font-weight:bold;vertical-align:top}
 +
    .tg .tg-yw4l{vertical-align:top}
 +
    </style>
 +
    <table class="tg">
 +
    <tr>
 +
      <th class="tg-9hbo">Host Strain</th>
 +
      <th class="tg-9hbo">Description</th>
 +
      <th class="tg-9hbo">Supplier</th>
 +
    </tr>
 +
    <tr>
 +
      <td class="tg-yw4l">CHO-DG44</td>
 +
      <td class="tg-yw4l">An immortal, adherent cell line derived from Chinese hamster (Cricetulus griseus) ovary cells. The strain we used is dihydrofolate reductase deficient.</td>
 +
      <td class="tg-yw4l">ATCC: CRL-9096 [10]</td>
 +
    </tr>
 +
    <tr>
 +
      <td class="tg-yw4l">AML-12</td>
 +
      <td class="tg-yw4l">An immortal, adherent cell line derived from mouse (Mus musculus) liver cells.</td>
 +
      <td class="tg-yw4l">ATCC: CRL-2254 [11]</td>
 +
    </tr>
 +
    </table>
 +
    </center>
 +
<div style='padding-top: 20px;'></div>
 +
    <div id="4"  style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Chemicals of Concern</b></div>
 +
    <div style='padding-top: 10px;'></div>
 +
 +
    <center>
 +
      <div style = 'line-height: 25px' >
 +
        Table 3. Chemicals of Concern
 +
    <p></p>
 +
      </div>
 +
      <style type="text/css">
 +
.tg  {border-collapse:collapse;border-spacing:0;}
 +
.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:black;}
 +
.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:black;}
 +
.tg .tg-j2xt{font-weight:bold;border-color:#002855;text-align:left;vertical-align:top}
 +
.tg .tg-0lax{text-align:left;vertical-align:top}
 +
</style>
 +
<table class="tg">
 +
  <tr>
 +
    <th class="tg-j2xt">Chemical name</th>
 +
    <th class="tg-j2xt">Occupational limit</th>
 +
    <th class="tg-j2xt">LD50</th>
 +
    <th class="tg-j2xt">Klamath Environmental concentration</th>
 +
    <th class="tg-j2xt">Cytotoxicology (In micromoles per liter)</th>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">Hydrogen Peroxide</td>
 +
    <td class="tg-0lax">75 mg/L</td>
 +
    <td class="tg-0lax">2000 mg/kg</td>
 +
    <td class="tg-0lax">--</td>
 +
    <td class="tg-0lax">Relevant dose is 60 uM</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">Warfarin</td>
 +
    <td class="tg-0lax">0.1 ug/L</td>
 +
    <td class="tg-0lax">374 mg/kgmice186 mg/kg rats</td>
 +
    <td class="tg-0lax">Qualitatively detected by Tom Young’s lab; unpublished data</td>
 +
    <td class="tg-0lax">Relevant dose is 0.0316 – 31.6 μM</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">2,4-D</td>
 +
    <td class="tg-0lax">10 ug/L</td>
 +
    <td class="tg-0lax">2019 ppm (min)12979 ppm(max)</td>
 +
    <td class="tg-0lax">0.424 - 7.49 ppm</td>
 +
    <td class="tg-0lax">-</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">Copper sulfate</td>
 +
    <td class="tg-0lax">1 ppb by inhalation</td>
 +
    <td class="tg-0lax">30 mg/kg</td>
 +
    <td class="tg-0lax">Copper measured at concentration of 19-67 ppm (112uM - 420uM)</td>
 +
    <td class="tg-0lax">Relevant dose is & lt;1.3ppm (6.25uM)</td>
 +
  </tr>
 +
  <tr>
 +
    <td class="tg-0lax">Metam Sodium</td>
 +
    <td class="tg-0lax">Limit of irritation 22 ug/L</td>
 +
    <td class="tg-0lax">781 mg/kg (rat)1836 ppm (min)</td>
 +
    <td class="tg-0lax">131309.21 kg</td>
 +
    <td class="tg-0lax">-</td>
 +
  </tr>
 
</table>
 
</table>
 
</center>
 
</center>
  
−
    <div style='padding-top: 30px;'></div>
 
−
    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VI. Plasmid</b></div>
 
  
−
      <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
+
<div style='padding-top: 20px;'></div>
−
        <p>We used pcDNA3.1(+) as our plasmid [18].</p>
+
<div id="5" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Plasmid</b></div>
−
    </div>
+
<div style='padding-top: 10px;'></div>
 +
<div style='padding-left:7%;padding-right: 7%'>
 +
  We used pcDNA3-EGFP as our plasmid [12]. The original plasmid is shown in Figure 1 below.
  
−
    <div style='padding-top: 30px;'></div>
+
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    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VII. Measurement</b></div>
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<div style='padding-top: 10px;'></div>
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  <div style = 'line-height: 25px' >
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    Figure 1. Map of pcDNA3-EGFP [13]
  
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  <img src="https://static.igem.org/mediawiki/2018/b/b4/T--UC_Davis--PM1c.png" alt="Plasmid map 1" style="width: 40%; margin-left: auto;margin-right: auto;" ;Padding: "0";>
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</center>
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<div style='padding-left:7%;padding-right: 7%'>
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  To prepare our constructs, we used restriction enzymes to remove the CMV enhancer, CMV promoter, and the T7 promoter. Promoter sequences were inserted using either restriction digest or Sequence and Ligation Independent Cloning (SLIC). Figure 2 below shows the complete plasmid for construct MT2_1.
  
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    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VIIII. Extenstion</b></div>
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    Figure 2. Map of pcDNA-EGFP_MT2_2 [13]
  
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          Our project opens up new opportunities for work with mammalian cells in the iGEM competition. By adding  ##  new mammalian parts to the registry, future teams will have the ability to easily access useful mammalian regulatory elements for use in their own constructs. Future teams may also benefit from our protocol for measuring fluorescence of adherent mammalian cells. One extension of our work is to transfect our construct into human cell lines. By using human cells, the bioassay will be a more accurate model for human health.
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  <img src="https://static.igem.org/mediawiki/2018/f/fa/T--UC_Davis--PM2.png" alt="Plasmid map 2" style="width: 40%; margin-left: auto;margin-right: auto;" ;Padding: "0";>
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    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>IX. References</b></div>
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<div id="6" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Measurement</b></div>
 +
<div style='padding-top: 10px;'></div>
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<div style='padding-left:7%;padding-right: 7%'>
 +
  Our bioassay was constructed on 96 well plates. Plates were seeded with 30,000 cells/well for AML12 cells and 20,000 cells/well for CHO cells and allowed to adhere for 24 hours. The cells were seeded such that 24 hours after reaching they would be at 70%-90% confluence. At this time, we transfected our cells with the construct containing the target promoter with reporter gene and allowed to sit for another 24 hours. Next, the complete growth media present in the wells were aspirated and replaced with clear media containing chemical of concern. We experimented with use of both a Tecan plate reader and a fluorescent microscope for measurement. For a full report on our measurement practices, please visit our  <a style ='color:  #d9a900;' href="https://2018.igem.org/Team:UC_Davis/Measurement">measurement page</a> and our  <a style ='color:  #d9a900;' href="https://2018.igem.org/Team:UC_Davis/Experiments">experiments page.</a>
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<div style='padding-top: 20px;'></div>
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        [1] “About EPA.” EPA, Environmental Protection Agency, 7 July 2018, www.epa.gov/aboutepa.
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<div id="7" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Extensions</b></div>
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      <p></p>
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<div style='padding-top: 10px;'></div>
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      [2] Johnson, David. “Superfund Sites: 1,317 US Spots Where Toxic Waste Was Dumped.” Time, Time Inc, 22 Mar. 2017, time.com/4695109/superfund-sites-toxic-waste-locations/.
+
<div style='padding-left:7%;padding-right: 7%'>
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      <p></p>
+
  Our project opens up new opportunities for work with mammalian cells in the iGEM competition. We have created comprehensive protocols for working with two mammalian cell lines, AML-12, and CHO-DG44, which may be downloaded on our Experiments Page. By adding new mammalian parts to the registry, future teams will have the ability to easily access useful mammalian regulatory elements for use in their own constructs. Future teams may also benefit from our protocol for measuring fluorescence of adherent mammalian cells. One extension of our work is to transfect our construct into human cell lines. By using human cells, the bioassay will be a more accurate model for human health.
−
      [3] Voosen, P. (2018). Wasteland. [online] Nationalgeographic.com. Available at: https://www.nationalgeographic.com/magazine/2014/12/superfund/ [Accessed 1 Aug. 2018].
+
</div>
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      <p></p>
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−
      [4] "UC Davis Superfund Research Program". UC Davis Superfund Research Program, 2018, https://www.superfund.ucdavis.edu/. Accessed 1 Aug 2018.
+
−
      <p></p>
+
−
      [5] “Ch. 21.15 Genetically Engineered Organisms | Yurok Tribal Code.” Yurok Tribe Tribal Code, Yurok Tribe, 10 Dec. 2015, yurok.tribal.codes/YTC/21.15.
+
−
      <p></p>
+
−
      [5] “Ch. 21.15 Genetically Engineered Organisms | Yurok Tribal Code.” Yurok Tribe Tribal Code, Yurok Tribe, 10 Dec. 2015, yurok.tribal.codes/YTC/21.15.
+
−
      <p></p>
+
−
      [6] Eagles-Smith, C.A., and B.L. Johnson, 2012, Contaminants in the Klamath Basin: Historical patterns, current distribution, and data gap identification: U.S. Geological Survey Administrative Report, 88 p.
+
−
      <p></p>
+
−
      [8] Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)
+
−
      Rudolf M. Lequin
+
−
      Clinical Chemistry Dec 2005, 51 (12) 2415-2418; DOI: 10.1373/clinchem.2005.051532
+
−
      <p></p>
+
−
      [9] Larochelle, Olivier & Labbé, Simon & Harrisson, Jean-François & Simard, Carl & Tremblay, Véronique & St-Gelais, Geneviève & Govindan, Manjapra Variath & Seguin, Carl. (2008). Nuclear Factor-1 and Metal Transcription Factor-1 Synergistically Activate the Mouse Metallothionein-1 Gene in Response to Metal Ions. The Journal of biological chemistry. 283. 8190-201. 10.1074/jbc.M800640200.
+
−
      <p></p>
+
−
      [10] Santos, Anderson K. et al. "Expression System Based On An Mtiia Promoter To Produce Hpsa In Mammalian Cell Cultures". Frontiers In Microbiology, vol 7, 2016. Frontiers Media SA, doi:10.3389/fmicb.2016.01280.
+
−
      <p></p>
+
−
      [11]  Li, Shuaizhang et al. "Functional Analysis Of The Dioxin Response Elements (Dres) Of The Murine CYP1A1 Gene Promoter: Beyond The Core DRE Sequence". International Journal Of Molecular Sciences, vol 15, no. 4, 2014, pp. 6475-6487. MDPI AG, doi:10.3390/ijms15046475.
+
−
      <p></p>
+
−
      [12] F.G. Schaap, A.E. Kremer, W.H. Lamers, P.L. Jansen, I.C. Gaemers. Fibroblast growth factor 21 is induced by endoplasmic reticulum stress
+
−
      Biochimie, 95 (2013), pp. 692-699, 10.1016/j.biochi.2012.10.019
+
−
      <p></p>
+
−
      [13]  Li, Dahui et al. "Genotoxic Evaluation Of The Insecticide Endosulfan Based On The Induced GADD153-GFP Reporter Gene Expression". Environmental Monitoring And Assessment, vol 176, no. 1-4, 2010, pp. 251-258. Springer Nature, doi:10.1007/s10661-010-1580-7.
+
−
      <p></p>
+
−
      [14]  Park, Jong Sung et al. "Isolation, Characterization And Chromosomal Localization Of The Human GADD153 Gene". Gene, vol 116, no. 2, 1992, pp. 259-267. Elsevier BV, doi:10.1016/0378-1119(92)90523-r.
+
−
      <p></p>
+
−
      [15] Mitra, Sumegha et al. "Gadd45a Promoter Regulation By A Functional Genetic Variant Associated With Acute Lung Injury". Plos ONE, vol 9, no. 6, 2014, p. e100169. Public Library Of Science (Plos), doi:10.1371/journal.pone.0100169
+
−
      <p></p>
+
−
      [16] Cell line available from ATCC at: https://www.atcc.org/Products/All/CRL-9096.aspx
+
−
      <p></p>
+
−
      [17] Cell line available from ATCC at: https://www.atcc.org/products/all/CRL-2254.aspx
+
−
      <p></p>
+
−
      [18] More detailed information regarding this plasmid is available from Addgene here: https://www.addgene.org/vector-database/2093/
+
  
 +
<center>
 +
    <div  style = 'padding-top:100px;padding-bottom: 60px; font-size: 30px; color: #d9a900'; ><b>Project Design Notebook </b></div>
 +
  </center>
 +
 +
</div>
 +
 +
The Project Design Notebook available for download below contains our thought and research as well as work space, as we designed our project. Within, we grapple with aspects of human practices, molecular biology, environmental toxicology, and the logistics of planning our experiments.
 +
 +
<div style='padding-top: 20px;'></div>
 +
<div id="8" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Project Design Notebook</b></div>
 +
 +
<center>
 +
  <embed src= "https://static.igem.org/mediawiki/2018/5/55/T--UC_Davis--PDN.pdf" width= "600" height= "500" style ='border-width: 3px;border-color: gray;'>
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  </center>
 +
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<div style='padding-top: 50px;'></div>
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<div style='padding-top: 20px;'></div>
 +
<div id="9" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>References</b></div>
 +
<div style='padding-top: 10px;'></div>
 +
<div style='padding-left:7%;padding-right: 7%'>
 +
  [1] “PDB101: Molecule of the Month: Green Fluorescent Protein (GFP).” PDB-101, RCSB PDB, June 2003, pdb101.rcsb.org/motm/42.
 +
      <p></p>
 +
      [2] Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)
 +
      Rudolf M. Lequin
 +
      Clinical Chemistry Dec 2005, 51 (12) 2415-2418; DOI: 10.1373/clinchem.2005.051532
 +
      <p></p>
 +
      [3]Kis Z, Pereira HSA, Homma T, Pedrigi RM, Krams R. 2015 Mammalian synthetic biology: emerging medical applications. J. R. Soc. Interface 12 : 20141000. http://dx.doi.org/10.1098/rsif.2014.1000
 +
      <p></p>
 +
      [4] Larochelle, Olivier & Labbé, Simon & Harrisson, Jean-François & Simard, Carl & Tremblay, Véronique & St-Gelais, Geneviève & Govindan, Manjapra Variath & Seguin, Carl. (2008). Nuclear Factor-1 and Metal Transcription Factor-1 Synergistically Activate the Mouse Metallothionein-1 Gene in Response to Metal Ions. The Journal of biological chemistry. 283. 8190-201. 10.1074/jbc.M800640200.
 +
      <p></p>
 +
[5] Santos, Anderson K. et al. "Expression System Based on An MtIIa Promoter to Produce Hpsa In Mammalian Cell Cultures". Frontiers in Microbiology, vol 7, 2016. Frontiers Media SA, doi:10.3389/fmicb.2016.01280.
 +
      <p></p>
 +
      [6] F.G. Schaap, A.E. Kremer, W.H. Lamers, P.L. Jansen, I.C. Gaemers. Fibroblast growth factor 21 is induced by endoplasmic reticulum stress
 +
      Biochimie, 95 (2013), pp. 692-699, 10.1016/j.biochi.2012.10.019
 +
      <p></p>
 +
      [7]  Li, Dahui et al. "Genotoxic Evaluation Of The Insecticide Endosulfan Based On The Induced GADD153-GFP Reporter Gene Expression". Environmental Monitoring And Assessment, vol 176, no. 1-4, 2010, pp. 251-258. Springer Nature, doi:10.1007/s10661-010-1580-7.
 +
      <p></p>
 +
      [8]  Park, Jong Sung et al. "Isolation, Characterization And Chromosomal Localization Of The Human GADD153 Gene". Gene, vol 116, no. 2, 1992, pp. 259-267. Elsevier BV, doi:10.1016/0378-1119(92)90523-r.
 +
      <p></p>
 +
      [9] Mitra, Sumegha et al. "Gadd45a Promoter Regulation By A Functional Genetic Variant Associated With Acute Lung Injury". Plos ONE, vol 9, no. 6, 2014, p. e100169. Public Library Of Science (Plos), doi:10.1371/journal.pone.0100169.
 +
      <p></p>
 +
      [10] Cell line available from ATCC at: https://www.atcc.org/Products/All/CRL-9096.aspx
 +
      <p></p>
 +
      [11] Cell line available from ATCC at: https://www.atcc.org/products/all/CRL-2254.aspx
 +
      <p></p>
 +
      [12] More detailed information regarding this plasmid is available from Addgene here: https://www.addgene.org/13031/ . pcDNA3-EGFP was a gift from Doug Golenbock (Addgene plasmid # 13031)
 +
      <p></p>
 +
      [13] Plasmid map created with software from SnapGene. Software is available at: snapgene.com
 +
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Latest revision as of 02:15, 18 October 2018

iGEM

Education
Bioassay Design Promoter Constructs Host Strains Chemicals of Concern Plasmid Measurement Extensions Project Design Notebook References
Project Design
Bioassay Design
We designed a mammalian cell-based bioassay that reports activation of specific stress pathways via fluorescence, for use in environmental toxicology. To do this, we selected transcriptionally regulated target genes which are present in mammalian cells and are involved in stress pathways. We isolated the promoters with transcription factor binding sites from these target genes and coupled them to a fluorescent reporter gene. We selected EGFP, a variant of green fluorescent protein (GFP). GFP is ubiquitous in synthetic biology due to its reliability and ease of measurement [1]. EGFP is derived from GFP and has been optimized for use in mammalian systems. When a chemical of concern is screened using our assay, if it triggers a specific stress response, the reporter gene will be expressed, causing the assay to fluoresce. The fluorescence of the assay can be quantitatively measured and analyzed. This assay will provide data on the effect of chemicals of concern on the physiological health of mammalian cells; measurements may be easily taken a range of concentrations, duration of exposure, salinities, pH, temperatures, nutrient availabilities, and other conditions. This also allows for measurement of synergistic or interfering effects due to multiple chemicals of concern present simultaneously.

We selected 8 promoter constructs derived from 5 target genes (see Figure 1 below) and coupled them to EGFP. This promoter and reporter gene construct were inserted into a plasmid and transfected into two cell lines (see Figure 2 below). The resulting bioassays were exposed to different chemicals of concern at a variety of concentrations and conditions (see Figure 3 below).

Why not use whole organisms?
A cell-based approach cannot replace in vivo toxicology studies. However, these studies require extensive funding, time, and other resources. By developing a relatively low-cost, cell-based bioassay, preliminary data may be quickly gathered, allowing for more informed decision making as to which in vivo studies are necessary. By using a cell-based preliminary assay, it is our hope that researchers will be able to quickly gather data, make more informed decisions, and save resources. Our cell-based bioassay may also be used to add to the body of knowledge concerning the effect of specific chemicals of concern on the physiological health of mammalian cells and the mechanism of stress.
Why not use cell-free biochemical assays, such as ELISA?
Antibody-based assays, such as Enzyme-Linked Immunosorbent Assay (ELISA), have been very successful in biomedical research, environmental toxicology, and other fields [2]. These cell-free methods involve selective binding of a target molecule to a prepared antibody. Such methods are very successful at identifying single, known compounds in an environmental sample, but do not provide any data regarding the effect of the chemical of concern on the health of a living cell. Similarly, the tools of analytical chemistry and organic chemistry may be used to great success when identifying molecules, but do not provide any data regarding the actual effect on a living cell.
Why use synthetic biology?
The goal of our bioassay is to create a tool that can be used to better understand the effect on the physiological health of mammalian cells of environmental toxins. An alternative way to achieve this knowledge is to expose cells to the chemicals of concern, lyse the cells, isolate the RNA, and run a quantitative-real-time-reverse-transcription-PCR in order to characterize and quantify the mRNAs present in the cell. However, this approach has limitations. It requires lysing the cells and cannot be used to gather real-time data about the behavior of the same cell over time. By using a fluorescent reporter gene, we can measure the induction of the reporter gene over time without lysing cells and can more easily take a large number of data points across different chemicals of concern, concentrations, and other variables. A fluorescent bioassay also reduces the amount of work required to measure many data points, compared to PCR based methods. Our bioassay also makes possible the future study of the behavior of a single cell over time after exposure to a chemical of concern, with the aid of microfluidics.
Why use mammalian cells?
We chose to use mammalian cells because they make much more accurate models for human health than bacteria or yeast. Furthermore, within the iGEM competition and the field of synthetic biology as a whole, there has been relatively little work with mammalian systems, compared to bacteria, yeast, and algae. Working with mammalian cells brings a variety of new challenges and opportunities to iGEM: they are more difficult and expensive to culture than bacteria, they require specialized equipment and safety training, they can be used to produce proteins suitable for use in human therapeutics (due to similar post-translational modifications), and they can be used for more complicated circuits and pathways utilizing spatial/temporal differentiation [3].
Why not use human cells?
Although human cell lines would make a superior model for human disease, compared to cell lines derived from hamsters and mice, for our project we chose not to use human cells. Work involving human cells requires specialized facilities, equipment, resources, and safety training. Human cells require a BSL-2 lab, which would have been more difficult for our team to use than our regular wetlab space, which is BSL-1. Additionally, as our project took place in the context of the iGEM competition, we wanted for other teams to be able to easily reproduce our findings and expand them. By using human cell lines, many teams, which lack access to a BSL-2 lab, would have had more difficulty in expanding upon our project. It would be relatively straightforward to insert our genetic constructs to a human cell line, and this presents an opportunity to extend our project.
Promoter Constructs
Table 1 shows the promoter constructs we used and the target genes from which they were derived. Full FASTA sequences for our promoters are available here.
Table 1. Promoter Constructs

Construct Target Gene Species of Origin Stress Pathway Size Further Reading
MT1 Metallothionein 1 Mus musculus Oxidative, heavy metal 305 nucleotides [4]
MT2_1 Metallothionein 2 Homo sapiens Oxidative, heavy metal 377 nucleotides [5]
MT2_2 Metallothionein 2 Homo sapiens Oxidative, heavy metal 59 nucleotides [5]
MT2_3 Metallothionein 2 Homo sapiens Oxidative, heavy metal 60 nucleotides; construct MT2_2 plus one base at 5’ end [5]
MT2_4 Metallothionein 2 Homo sapiens Oxidative, heavy metal 60 nucleotides; construct MT2_2 plus one base at 3’ end [5]
FGF FGF21 Homo sapiens Unfolded protein response (UPR), endoplasmic reticulum stress 695 nucleotides [6]
GD153 GADD153 Cricetulus griseus Organochlorine biocides, genotoxins 811 nucleotides [7], [8]
GD45 GADD45α Homo sapiens Genotoxins, mechanical stress 1006 nucleotides [9]
Host Strains
Table 2. Host Strains

Host Strain Description Supplier
CHO-DG44 An immortal, adherent cell line derived from Chinese hamster (Cricetulus griseus) ovary cells. The strain we used is dihydrofolate reductase deficient. ATCC: CRL-9096 [10]
AML-12 An immortal, adherent cell line derived from mouse (Mus musculus) liver cells. ATCC: CRL-2254 [11]
Chemicals of Concern
Table 3. Chemicals of Concern

Chemical name Occupational limit LD50 Klamath Environmental concentration Cytotoxicology (In micromoles per liter)
Hydrogen Peroxide 75 mg/L 2000 mg/kg -- Relevant dose is 60 uM
Warfarin 0.1 ug/L 374 mg/kgmice186 mg/kg rats Qualitatively detected by Tom Young’s lab; unpublished data Relevant dose is 0.0316 – 31.6 μM
2,4-D 10 ug/L 2019 ppm (min)12979 ppm(max) 0.424 - 7.49 ppm -
Copper sulfate 1 ppb by inhalation 30 mg/kg Copper measured at concentration of 19-67 ppm (112uM - 420uM) Relevant dose is & lt;1.3ppm (6.25uM)
Metam Sodium Limit of irritation 22 ug/L 781 mg/kg (rat)1836 ppm (min) 131309.21 kg -
Plasmid
We used pcDNA3-EGFP as our plasmid [12]. The original plasmid is shown in Figure 1 below.
Figure 1. Map of pcDNA3-EGFP [13]

Plasmid map 1
To prepare our constructs, we used restriction enzymes to remove the CMV enhancer, CMV promoter, and the T7 promoter. Promoter sequences were inserted using either restriction digest or Sequence and Ligation Independent Cloning (SLIC). Figure 2 below shows the complete plasmid for construct MT2_1.
Figure 2. Map of pcDNA-EGFP_MT2_2 [13]

Plasmid map 2
Measurement
Our bioassay was constructed on 96 well plates. Plates were seeded with 30,000 cells/well for AML12 cells and 20,000 cells/well for CHO cells and allowed to adhere for 24 hours. The cells were seeded such that 24 hours after reaching they would be at 70%-90% confluence. At this time, we transfected our cells with the construct containing the target promoter with reporter gene and allowed to sit for another 24 hours. Next, the complete growth media present in the wells were aspirated and replaced with clear media containing chemical of concern. We experimented with use of both a Tecan plate reader and a fluorescent microscope for measurement. For a full report on our measurement practices, please visit our measurement page and our experiments page.
Extensions
Our project opens up new opportunities for work with mammalian cells in the iGEM competition. We have created comprehensive protocols for working with two mammalian cell lines, AML-12, and CHO-DG44, which may be downloaded on our Experiments Page. By adding new mammalian parts to the registry, future teams will have the ability to easily access useful mammalian regulatory elements for use in their own constructs. Future teams may also benefit from our protocol for measuring fluorescence of adherent mammalian cells. One extension of our work is to transfect our construct into human cell lines. By using human cells, the bioassay will be a more accurate model for human health.
Project Design Notebook
The Project Design Notebook available for download below contains our thought and research as well as work space, as we designed our project. Within, we grapple with aspects of human practices, molecular biology, environmental toxicology, and the logistics of planning our experiments.
Project Design Notebook
References
[1] “PDB101: Molecule of the Month: Green Fluorescent Protein (GFP).” PDB-101, RCSB PDB, June 2003, pdb101.rcsb.org/motm/42.

[2] Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA) Rudolf M. Lequin Clinical Chemistry Dec 2005, 51 (12) 2415-2418; DOI: 10.1373/clinchem.2005.051532

[3]Kis Z, Pereira HSA, Homma T, Pedrigi RM, Krams R. 2015 Mammalian synthetic biology: emerging medical applications. J. R. Soc. Interface 12 : 20141000. http://dx.doi.org/10.1098/rsif.2014.1000

[4] Larochelle, Olivier & Labbé, Simon & Harrisson, Jean-François & Simard, Carl & Tremblay, Véronique & St-Gelais, Geneviève & Govindan, Manjapra Variath & Seguin, Carl. (2008). Nuclear Factor-1 and Metal Transcription Factor-1 Synergistically Activate the Mouse Metallothionein-1 Gene in Response to Metal Ions. The Journal of biological chemistry. 283. 8190-201. 10.1074/jbc.M800640200.

[5] Santos, Anderson K. et al. "Expression System Based on An MtIIa Promoter to Produce Hpsa In Mammalian Cell Cultures". Frontiers in Microbiology, vol 7, 2016. Frontiers Media SA, doi:10.3389/fmicb.2016.01280.

[6] F.G. Schaap, A.E. Kremer, W.H. Lamers, P.L. Jansen, I.C. Gaemers. Fibroblast growth factor 21 is induced by endoplasmic reticulum stress Biochimie, 95 (2013), pp. 692-699, 10.1016/j.biochi.2012.10.019

[7] Li, Dahui et al. "Genotoxic Evaluation Of The Insecticide Endosulfan Based On The Induced GADD153-GFP Reporter Gene Expression". Environmental Monitoring And Assessment, vol 176, no. 1-4, 2010, pp. 251-258. Springer Nature, doi:10.1007/s10661-010-1580-7.

[8] Park, Jong Sung et al. "Isolation, Characterization And Chromosomal Localization Of The Human GADD153 Gene". Gene, vol 116, no. 2, 1992, pp. 259-267. Elsevier BV, doi:10.1016/0378-1119(92)90523-r.

[9] Mitra, Sumegha et al. "Gadd45a Promoter Regulation By A Functional Genetic Variant Associated With Acute Lung Injury". Plos ONE, vol 9, no. 6, 2014, p. e100169. Public Library Of Science (Plos), doi:10.1371/journal.pone.0100169.

[10] Cell line available from ATCC at: https://www.atcc.org/Products/All/CRL-9096.aspx

[11] Cell line available from ATCC at: https://www.atcc.org/products/all/CRL-2254.aspx

[12] More detailed information regarding this plasmid is available from Addgene here: https://www.addgene.org/13031/ . pcDNA3-EGFP was a gift from Doug Golenbock (Addgene plasmid # 13031)

[13] Plasmid map created with software from SnapGene. Software is available at: snapgene.com
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UC Davis iGEM 2018