Team:ITESM-CEM/Project/Experiments
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<sub2><a href="#One" style="color: #FFF;">PCR's</a></sub2> | <sub2><a href="#One" style="color: #FFF;">PCR's</a></sub2> | ||
<sub2><a href="#Two" style="color: #FFF;">Mammalian Cell Transfection</a></sub2> | <sub2><a href="#Two" style="color: #FFF;">Mammalian Cell Transfection</a></sub2> | ||
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<sub2><a href="#Four" style="color: #FFF;">Protein Expression</a></sub2> | <sub2><a href="#Four" style="color: #FFF;">Protein Expression</a></sub2> | ||
<sub2><a href="#Five" style="color: #FFF;">NeoR Characterization</a></sub2> | <sub2><a href="#Five" style="color: #FFF;">NeoR Characterization</a></sub2> | ||
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<p style="text-align: justify; text-justify: inter-word;">pcDNA 3.1 Myc-His A is an expression plasmid that contains BGHPA, a constitutive promoter and an origin of replication that work in mammalian cells. To see if the plasmid was working correctly, a cassette was expressed with GFP to see if the plasmid worked in a specific eukaryotic cell line (Marc145), so the enzyme could be inserted and the analysis of functionality of the enzyme by substrate degradation could be started. </p><br> | <p style="text-align: justify; text-justify: inter-word;">pcDNA 3.1 Myc-His A is an expression plasmid that contains BGHPA, a constitutive promoter and an origin of replication that work in mammalian cells. To see if the plasmid was working correctly, a cassette was expressed with GFP to see if the plasmid worked in a specific eukaryotic cell line (Marc145), so the enzyme could be inserted and the analysis of functionality of the enzyme by substrate degradation could be started. </p><br> | ||
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<p><pie><b>Image 1.</b>Cells before (left) and after (right) transfection with plasmid pcDNA 3.1 Myc-His A with 7-dehydratase gene using lipofectamine as a transfecting agent. </p></pie> | <p><pie><b>Image 1.</b>Cells before (left) and after (right) transfection with plasmid pcDNA 3.1 Myc-His A with 7-dehydratase gene using lipofectamine as a transfecting agent. </p></pie> | ||
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<p style="text-align: justify; text-justify: inter-word;">As fluorescence of this enzyme cannot be detected, its characterization is going to be determined by an antibiotic resistance at a certain concentration (Neomycin), where cells are able to keep dividing, except for the ones without the resistance gene.</p> | <p style="text-align: justify; text-justify: inter-word;">As fluorescence of this enzyme cannot be detected, its characterization is going to be determined by an antibiotic resistance at a certain concentration (Neomycin), where cells are able to keep dividing, except for the ones without the resistance gene.</p> | ||
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<gotop><a href="#top">Back to top ↑</a></gotop><br><br> | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | ||
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Both groups consisted on thirteen essay tubes with 5 ml of LB media each one with a different concentration on Neomycin as shown in table 1 and 2. The tubes were cultured on a shaker for 18 hours at 250 rpm/37ºC. Afterwards, each tube had its optical density measured at 600 nm using as LB media as a blank with the same antibiotic concentration. Five neomycin concentrations were chosen to perform petri dish cultures but only with the trouble group to perform a C.F.U. count. | Both groups consisted on thirteen essay tubes with 5 ml of LB media each one with a different concentration on Neomycin as shown in table 1 and 2. The tubes were cultured on a shaker for 18 hours at 250 rpm/37ºC. Afterwards, each tube had its optical density measured at 600 nm using as LB media as a blank with the same antibiotic concentration. Five neomycin concentrations were chosen to perform petri dish cultures but only with the trouble group to perform a C.F.U. count. | ||
</p> | </p> | ||
+ | <p><pie><b>Table 1.</b> Control group without neomycin</p></pie><br> | ||
+ | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/a/a7/ControlTable.jpg" height="366" width="700" align="middle" hspace="10" BORDER=10><br></p><br> | ||
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+ | <p><pie><b>Table 2.</b> Positive Group with neomycin</p></pie><br> | ||
+ | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/1/1d/NeoR_positivo.jpg" height="411" width="700" align="middle" hspace="10" BORDER=10><br></p><br> | ||
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<gotop><a href="#top">Back to top ↑</a></gotop><br><br> | <gotop><a href="#top">Back to top ↑</a></gotop><br><br> | ||
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Latest revision as of 03:54, 18 October 2014
ITESM-CEM | Enzy7-K me |
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