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

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]

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]

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

iGEM-Footer

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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. Bioassay Design</b></div>
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-      <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
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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
+    color: #d9a900;
-        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 [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
-        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
-        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
-        or interfering effects due to multiple chemicals of concern present simultaneously.
-        <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).
-        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?
-        </b></i>
-        </div>
-  <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
-       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>
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+}
-            Why not use cell-free biochemical assays, such as ELISA?
-          </b></i>
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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?
-        </b></i>
-        </div>
-  <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
+.parentContent {
-         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
+   position: relative;
-        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
+}
-        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.
-   </div>
-  <div style='padding-top: 20px;'></div>
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-          Why use mammalian cells?
-        </b></i>
-        </div>
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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
+   </style>
-        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
+   <body>
-        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
-        differentiation, and they are much more sensitive to chemicals in the environment (allowing for more sensitive biosensors and bioassays).
-   </div>
-   <div style='padding-top: 20px;'></div>
-        <div style = 'font-size: 20px;color: #a6d4f4; padding-right: 130PX; padding-left: 130PX;line-height: 25px; padding-bottom: 20px;' ><b><i>
-          Why not use human cells?
-        </b></i>
-        </div>
-       <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
+    <div class="parentContent">
-      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,
+       <div class="sidenav" style='padding-top: 250px;' >
-         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
+        <a href="#1">Bioassay Design</a>
-         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
+         <a href="#2">Promoter Constructs</a>
-         be relatively straightforward to insert our genetic constructs to a human cell line, and this presents an opportunity to extend our project.
+        <a href="#3">Host Strains</a>
+        <a href="#4">Chemicals of Concern</a>
+         <a href="#5">Plasmid</a>
+         <a href="#6">Measurement</a>
+        <a href="#7">Extensions</a>
+        <a href="#8">Project Design Notebook</a>
+        <a href="#9">References</a>
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+    <div  style = 'padding-top:100px;padding-bottom: 60px; font-size: 30px; color: #d9a900'; ><b>Project Design </b></div>
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      <div style='padding-top: 30px;'></div>
-     <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>II. Promoter Constructs</b></div>
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-      <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
+    <div id="1" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Bioassay Design</b></div>
+    <div style='padding-top: 10px;'></div>
+    <div style='padding-left:7%;padding-right: 7%'>
+      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.
-        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 < <a href="https://2018.igem.org/Team:UC_Davis/SupplementalMaterials">here</a>.
+<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).
+</p>
+</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 whole organisms?</i></b></div>
-      </div>
+<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>
-<center>
+<div style='padding-top: 10px;'></div>
-  <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' >
+<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>
-    Figure 2. Promoter Constructs
+<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%'>
+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>
+<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-left:10%;padding-right: 7%'>
+  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>
    <style type="text/css">
 .tg  {border-collapse:collapse;border-spacing:0;border-color:#aaa;}
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@@ Line 128: / Line 172: @@
    <td class="tg-yw4l">Oxidative, heavy metal</td>
    <td class="tg-yw4l">305 nucleotides</td>
-   <td class="tg-yw4l">[9]</td>
+   <td class="tg-yw4l">[4]</td>
 </tr>
 <tr>
@@ Line 136: / Line 180: @@
    <td class="tg-yw4l">Oxidative, heavy metal</td>
    <td class="tg-yw4l">377 nucleotides</td>
-   <td class="tg-yw4l">[10]</td>
+   <td class="tg-yw4l">[5]</td>
 </tr>
 <tr>
@@ Line 144: / Line 188: @@
    <td class="tg-yw4l">Oxidative, heavy metal</td>
    <td class="tg-yw4l">59 nucleotides</td>
-   <td class="tg-yw4l">[10]</td>
+   <td class="tg-yw4l">[5]</td>
 </tr>
 <tr>
@@ Line 152: / Line 196: @@
    <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">[10]</td>
+   <td class="tg-yw4l">[5]</td>
 </tr>
 <tr>
@@ Line 160: / Line 204: @@
    <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">[10]</td>
+   <td class="tg-yw4l">[5]</td>
-</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">Dioxins, organochlorine biocides</td>
-  <td class="tg-yw4l">1902 nucleotides</td>
-  <td class="tg-yw4l">[11]</td>
 </tr>
 <tr>
@@ Line 176: / Line 212: @@
    <td class="tg-yw4l">Unfolded protein response (UPR), endoplasmic reticulum stress</td>
    <td class="tg-yw4l">695 nucleotides</td>
-   <td class="tg-yw4l">[12]</td>
+   <td class="tg-yw4l">[6]</td>
 </tr>
 <tr>
@@ Line 184: / Line 220: @@
    <td class="tg-yw4l">Organochlorine biocides, genotoxins</td>
    <td class="tg-yw4l">811 nucleotides</td>
-   <td class="tg-yw4l">[13], [14]</td>
+   <td class="tg-yw4l">[7], [8]</td>
 </tr>
 <tr>
@@ Line 192: / Line 228: @@
    <td class="tg-yw4l">Genotoxins, mechanical stress</td>
    <td class="tg-yw4l">1006 nucleotides</td>
-   <td class="tg-yw4l">[15]</td>
+   <td class="tg-yw4l">[9]</td>
 </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>
-     <div style='padding-top: 30px;'></div>
+      <style type="text/css">
-     <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>III. Host Strains</b></div>
+     .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>
+    <center>
-  <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' >
+      <div style = 'line-height: 25px' >
-    Figure 3: Host Strains
+        Table 3. Chemicals of Concern
+    <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;}
-.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:#aaa;color:#333;background-color:#fff;}
+.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:#aaa;color:#fff;background-color:#002855;}
+.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-9hbo{font-weight:bold;vertical-align:top}
+.tg .tg-j2xt{font-weight:bold;border-color:#002855;text-align:left;vertical-align:top}
-.tg .tg-yw4l{vertical-align:top}
+.tg .tg-0lax{text-align:left;vertical-align:top}
 </style>
 <table class="tg">
-<tr>
+  <tr>
-  <th class="tg-9hbo">Host Strain</th>
+    <th class="tg-j2xt">Chemical name</th>
-  <th class="tg-9hbo">Description</th>
+    <th class="tg-j2xt">Occupational limit</th>
-  <th class="tg-9hbo">Supplier</th>
+    <th class="tg-j2xt">LD50</th>
-</tr>
+    <th class="tg-j2xt">Klamath Environmental concentration</th>
-<tr>
+    <th class="tg-j2xt">Cytotoxicology (In micromoles per liter)</th>
-   <td class="tg-yw4l">CHO-DG44</td>
+  </tr>
-   <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>
+  <tr>
-  <td class="tg-yw4l">ATCC: CRL-9096 [16]</td>
+    <td class="tg-0lax">Hydrogen Peroxide</td>
-</tr>
+    <td class="tg-0lax">75 mg/L</td>
-<tr>
+    <td class="tg-0lax">2000 mg/kg</td>
-  <td class="tg-yw4l">AML-12</td>
+    <td class="tg-0lax">--</td>
-  <td class="tg-yw4l">An immortal, adherent cell line derived from mouse (Mus musculus) liver cells.</td>
+    <td class="tg-0lax">Relevant dose is 60 uM</td>
-   <td class="tg-yw4l">ATCC: CRL-2254 [17]</td>
+   </tr>
-</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>
 </center>
+<div style='padding-top: 20px;'></div>
+<div id="5" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Plasmid</b></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>
+<div style='padding-top: 10px;'></div>
+<center>
+  <div style = 'line-height: 25px' >
+    Figure 1. Map of pcDNA3-EGFP [13]
-    <div style='padding-top: 30px;'></div>
+<p></p>
-    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>IV.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">
+  <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";>
-.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>
-  <th class="tg-9hbo">Chemical of Concern</th>
-  <th class="tg-9hbo">Class</th>
-</tr>
-<tr>
-  <td class="tg-yw4l">Copper Sulfate</td>
-  <td class="tg-yw4l">Heavy metal</td>
-</tr>
-<tr>
-  <td class="tg-yw4l">Zinc Sulfate</td>
-  <td class="tg-yw4l">Heavy metal</td>
-</tr>
-<tr>
-  <td class="tg-yw4l">Metam Sodium</td>
-  <td class="tg-yw4l">Organosulfur biocide</td>
-</tr>
-<tr>
-  <td class="tg-yw4l">2,4-D</td>
-  <td class="tg-yw4l">Organochlorine herbicide</td>
-</tr>
-<tr>
-  <td class="tg-yw4l">Warfarin</td>
-  <td class="tg-yw4l">Pharmaceutical anticoagulant, pesticide</td>
-</tr>
-<tr>
-  <td class="tg-yw4l">Hydrogen Peroxide</td>
-  <td class="tg-yw4l">Oxidizing agent</td>
-</tr>
-</table>
 </center>
+<div style='padding-left:7%;padding-right: 7%'>
+  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.
-    <div style='padding-top: 30px;'></div>
+</div>
-    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>V. Plasmid</b></div>
-      <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
-        We used pcDNA3-EGFP as our plasmid [18]. The original plasmid is shown in Figure 5 below.
-    </div>
 <center>
-      <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' >
+  <div style = 'line-height: 25px' >
-        Figure 5. Map of pcDNA3-EGFP [19]
+    Figure 2. Map of pcDNA-EGFP_MT2_2 [13]
-      </div>
-    <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";>
-</center>
-    <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
-      To prepare our constructs, we used restriction enzymes to remove the CMV enhancer, CMV promoter, and the T7 promoter. Promoter sequences were inserted using Sequence and Ligation Independent Cloning (SLIC). Figure 6 below shows the complete plasmid for construct MT2_1.
+<p></p>
    </div>
-   <center>
+   <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";>
-        <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' >
-          Figure 6. Map of pcDNA-EGFP_MT2_1 [19]
-        </div>
-    <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";>
 </center>
-    <div style='padding-top: 30px;'></div>
-    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VI. Measurement</b></div>
-      <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
+<div style='padding-top: 20px;'></div>
-        <p></p>
+<div id="6" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Measurement</b></div>
-    </div>
+<div style='padding-top: 10px;'></div>
+<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>
+</div>
-    <div style='padding-top: 30px;'></div>
+<div style='padding-top: 20px;'></div>
-    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VII. Extension</b></div>
+<div id="7" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Extensions</b></div>
+<div style='padding-top: 10px;'></div>
+<div style='padding-left:7%;padding-right: 7%'>
+  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.
+</div>
-      <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' >
+<center>
+    <div  style = 'padding-top:100px;padding-bottom: 60px; font-size: 30px; color: #d9a900'; ><b>Project Design Notebook </b></div>
+  </center>
-          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.
+</div>
-    </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: 30px;'></div>
+<div style='padding-top: 20px;'></div>
-    <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VIII. References</b></div>
+<div id="8" style = 'padding-left:7%;padding-bottom: 22px; font-size: 25px; color: #667d9d'; ><b>Project Design Notebook</b></div>
-      <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' >
+<center>
-        [1] “About EPA.” EPA, Environmental Protection Agency, 7 July 2018, www.epa.gov/aboutepa.
+  <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;'>
-      <p></p>
+  </center>
-      [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/.
-      <p></p>
-      [3] Voosen, P. (2018). Wasteland. [online] Nationalgeographic.com. Available at: https://www.nationalgeographic.com/magazine/2014/12/superfund/ [Accessed 1 Aug. 2018].
-      <p></p>
-      [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/
+<div style='padding-top: 50px;'></div>
+<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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