The Experiments

If you choose to create a model during your project, please write about it here. Modeling is not an essential part of iGEM, but we encourage any and all teams to model some aspect of their project. See previous "Best Model" awards for more information.

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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.

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.

Procedure

The characterization was made using two groups: the trouble group and the control group. The trouble group was made using an E.coli DH5-α inoculum, transformed with the NeoR gene inserted in psB1C3 using the constitutive promoter BBa_K823012. The control group used an untransformed E.coli DH5-α inoculum.

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. Afterwards each tube had its optical density measured at 600 nm using as blank LB media at 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.