Team:ITESM-CEM/Project/Experiments
From 2014.igem.org
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<p> The scope of our Project is to express in mammalian cells the new synthetic pathway able to metabolize 7-ketocholesterol. Just like bacteria, mammalian cells also need a selective gene to identify successfully transformed organisms. NeoR is a gene that encodes an aminoglycoside 3'-phosphotransferase enzyme, which provides in theory a resistance to Neomycin and its derivatives. The antibiotic that will be used to select the successfully transformed mammalian cells is G418®, a Neomycin derivative which only affects mammalian cells. <br><br> | <p> The scope of our Project is to express in mammalian cells the new synthetic pathway able to metabolize 7-ketocholesterol. Just like bacteria, mammalian cells also need a selective gene to identify successfully transformed organisms. NeoR is a gene that encodes an aminoglycoside 3'-phosphotransferase enzyme, which provides in theory a resistance to Neomycin and its derivatives. The antibiotic that will be used to select the successfully transformed mammalian cells is G418®, a Neomycin derivative which only affects mammalian cells. <br><br> | ||
- | However the scope of our project exceeded the possibilities given the time constrains. Therefore a characterization in a cell culture was not done due to time limitations. The NeoR gene was characterized using an E.coli culture and Neomycin as selective antibiotic. The NeoR gene was obtained by PCR from pcDNA3.1myc his A, and following iGEM instructions, the gene was introduced in the plasmid psB1C3 as it is shown in the following picture. | + | However the scope of our project exceeded the possibilities given the time constrains. Therefore a characterization in a cell culture was not done due to time limitations. The NeoR gene was characterized using an E.coli culture and Neomycin as selective antibiotic. The NeoR gene was obtained by PCR from pcDNA3.1myc his A, and following iGEM instructions, the gene was introduced in the plasmid psB1C3 as it is shown in the following picture.<br><br> |
- | <img src="https://2014.igem.org/File:Plasmido_BBa_K1313004.jpg" align="right" width="569" height="569" hspace="20"> | + | <p><img src="https://2014.igem.org/File:Plasmido_BBa_K1313004.jpg" align="right" width="569" height="569" hspace="20"></p> |
<h4>Procedure</h4> | <h4>Procedure</h4> |
Revision as of 06:11, 17 October 2014
ITESM-CEM | Enzy7-K me |
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