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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. | 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> | ||
+ | <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 cell-free biochemical assays, such as ELISA? | ||
+ | |||
+ | </b></i> | ||
+ | </div> | ||
+ | <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' > | ||
+ | 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. | ||
+ | </div> | ||
<div style='padding-top: 20px;'></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> | <div style = 'font-size: 20px;color: #a6d4f4; padding-right: 130PX; padding-left: 130PX;line-height: 25px; padding-bottom: 20px;' ><b><i> | ||
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<div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' > | <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' > | ||
− | <p></p> | + | <p> |
+ | 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 . | ||
+ | |||
+ | </p> | ||
</div> | </div> | ||
+ | <center> | ||
+ | <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' > | ||
+ | Figure 2. Promoter Constructs | ||
+ | </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:10px 5px;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:#f38630;} | ||
+ | .tg .tg-9hbo{font-weight:bold;vertical-align:top} | ||
+ | .tg .tg-yw4l{vertical-align:top} | ||
+ | </style> | ||
+ | <table class="tg"> | ||
+ | <tr> | ||
+ | <th class="tg-9hbo">Construct</th> | ||
+ | <th class="tg-9hbo">Target Gene</th> | ||
+ | <th class="tg-9hbo">Species of Origin</th> | ||
+ | <th class="tg-9hbo">Description</th> | ||
+ | <th class="tg-9hbo">Further Reading</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">MT1</td> | ||
+ | <td class="tg-yw4l">Metallothionein 1</td> | ||
+ | <td class="tg-yw4l">Mus musculus</td> | ||
+ | <td class="tg-yw4l">//</td> | ||
+ | <td class="tg-yw4l">[9]</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">MT2_1</td> | ||
+ | <td class="tg-yw4l">Metallothionein 2</td> | ||
+ | <td class="tg-yw4l">Homo sapiens</td> | ||
+ | <td class="tg-yw4l">full</td> | ||
+ | <td class="tg-yw4l">[10]</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">MT2_2</td> | ||
+ | <td class="tg-yw4l">Metallothionein 2</td> | ||
+ | <td class="tg-yw4l">Homo sapiens</td> | ||
+ | <td class="tg-yw4l">59</td> | ||
+ | <td class="tg-yw4l">[10]</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">MT2_3</td> | ||
+ | <td class="tg-yw4l">Metallothionein 2</td> | ||
+ | <td class="tg-yw4l">Homo sapiens</td> | ||
+ | <td class="tg-yw4l">60 first A</td> | ||
+ | <td class="tg-yw4l">[10]</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-yw4l">MT2_4</td> | ||
+ | <td class="tg-yw4l">Metallothionein 2</td> | ||
+ | <td class="tg-yw4l">Homo sapiens</td> | ||
+ | <td class="tg-yw4l">60 last A</td> | ||
+ | <td class="tg-yw4l">[10]</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">//</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-top: 30px;'></div> | <div style='padding-top: 30px;'></div> | ||
− | <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>IV. | + | <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>IV. Host Strains</b></div> |
− | + | <center> | |
− | + | <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' > | |
− | + | Figure 3: Host Strains | |
+ | |||
+ | </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:10px 5px;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 .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 [16]</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 [17]</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
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<div style='padding-top: 30px;'></div> | <div style='padding-top: 30px;'></div> | ||
− | <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>V. | + | <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> | ||
+ | <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-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;' > | ||
+ | <p>We used pcDNA3.1(+) as our plasmid [18].</p> | ||
+ | </div> | ||
+ | |||
+ | <div style='padding-top: 30px;'></div> | ||
+ | <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VII. Measurement</b></div> | ||
<div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' > | <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' > | ||
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</div> | </div> | ||
+ | <div style='padding-top: 30px;'></div> | ||
+ | <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>VIIII. Extenstion</b></div> | ||
+ | <div style = 'padding-right: 130PX; padding-left: 130PX; text-indent: 50px;line-height: 25px;' > | ||
+ | <p> | ||
+ | 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. | ||
+ | </p> | ||
+ | </div> | ||
<div style='padding-top: 30px;'></div> | <div style='padding-top: 30px;'></div> | ||
− | <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b> | + | <div style = 'padding-left: 130PX; padding-bottom: 22px; font-size: 25px; color: #d9a900'; ><b>IX. References</b></div> |
− | + | ||
− | <div style = 'padding-right: 130PX; padding-left: 130PX | + | <div style = 'padding-right: 130PX; padding-left: 130PX;line-height: 25px' > |
+ | [1] “About EPA.” EPA, Environmental Protection Agency, 7 July 2018, www.epa.gov/aboutepa. | ||
<p></p> | <p></p> | ||
+ | [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> | </div> | ||
+ | <div style='padding-top: 100px;'></div> | ||
+ | |||
+ | </body> | ||
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{{Template: UC_Davis/footer}} | {{Template: UC_Davis/footer}} |
Revision as of 17:46, 8 August 2018
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; 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.
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.
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.
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, and the significance of the wild salmon to the tribe’s cultural values.
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.
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.
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.
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 cells.
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).
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 [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.
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 .
Construct | Target Gene | Species of Origin | Description | Further Reading |
---|---|---|---|---|
MT1 | Metallothionein 1 | Mus musculus | // | [9] |
MT2_1 | Metallothionein 2 | Homo sapiens | full | [10] |
MT2_2 | Metallothionein 2 | Homo sapiens | 59 | [10] |
MT2_3 | Metallothionein 2 | Homo sapiens | 60 first A | [10] |
MT2_4 | Metallothionein 2 | Homo sapiens | 60 last A | [10] |
CYP | CYP1A1 | Mus musculus | // | [11] |
FGF | FGF21 | Homo sapiens | // | [12] |
GD153 | GADD153 | Cricetulus griseus | // | [13], [14] |
GD45 | GADD45α | Homo sapiens | // | [15] |
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 [16] |
AML-12 | An immortal, adherent cell line derived from mouse (Mus musculus) liver cells. | ATCC: CRL-2254 [17] |
Chemical of Concern | Class |
---|---|
Copper Sulfate | Heavy metal |
Zinc Sulfate | Heavy metal |
Metam Sodium | Organosulfur biocide |
2,4-D | Organochlorine herbicide |
Warfarin | Pharmaceutical anticoagulant, pesticide |
Hydrogen Peroxide | Oxidizing agent |
We used pcDNA3.1(+) as our plasmid [18].
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.