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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Each protein was inserted in Escherichia coli through pPROEX B which is a bacterial expression plasmid. This plasmid was used due to its characteristics, which include TRC promoter inducible by IPTG and has a 6x histidine tag in N-terminal end.

In the case of cholesterol oxidase, the enzyme was successfully introduced in the plasmid mentioned before. The protein wasn’t overexpressed by induction with IPTG, so no further analysis was made because this enzyme is well characterized and there is more information about it in BRENDA Enzymes as EC 1.1.3.6 – cholesterol oxidase, Chromobacterium sp.

Oxoacyl reductase was analyzed by SDS-PAGE in a 15% acrylamide gel. First, main cultures of the different colonies grown in the plate were inoculated in 40 ml of LB with ampicillin (100 ug/ml). After a few hours in the shaker, optical density was measured repeatedly until it was between 0.5 and 0.65, considering this measurement our time zero. Right after it, IPTG 1 mM was added to start the overexpression of the protein. Each hour, the absorbance of each culture was registered until the time six.

Table 1. Different absorbances measured each hour of the cultures of oxoacyl reductase.

The samples taken each hour were centrifuged 5 min/13500 rpm to concentrate the pellet in the bottom of the microtube. The supernatant was thrown into a waste glass, and the pellets were resuspended in different volumes of Laemmli buffer, depending on the value of absorbance obtained. The criteria used to determine the volume of buffer, was taking into consideration the absorbance of the time zero, which would represent the 100 percent of buffer added (100 ul in this sample), and going up or down depending of the absorbances of the other samples. The different volumes are now shown.

Table 2. Volumes of Laemmli buffer depending of the absorbance value of each sample.

The samples were loaded in a 15% acrylamide gel, using Precision Plus Protein TM Dual Color Standards, for 20 minutes/90 V for the stacking gel and 60 minutes/150V for the resolving gel. The results are now presented:

Only the samples shown in the image before were the ones that presented notable bands that represent our protein of interest. As expected, the most remarked band is the one of the time 3, which means that inductions was taken correctly and more protein was produced, in other words, the protein was overexpressing. The band marked with the arrow represents a protein that weights approximately 34 kDa, which corresponds to the molecular weight of oxoacyl reductase according to ExPASy’s Compute pI/MW tool.

7-dehydratase was analyzed by SDS-PAGE in a 15% acrylamide gel using Precision Plus Protein TM Unstained Standards, for 20 minutes/90 V for the stacking gel and 90 minutes/110V for the resolving gel. Four samples were taken, including one before and after induction with IPTG, one from the soluble phase and one from the inclusion bodies; all prepared with Laemmli buffer. The results are shown in the image below.

No analysis of solubility was realized due to the quantity of protein. It was supposed to be done exactly the same than oxoacyl reductase, as the protein was found in a notable way in the inclusion bodies as shown in the lane 5.

For both enzymes no further work was done. After the identification of each of them, and after the analysis of solubility, the proteins have to be purified by affinity chromatography with a Invitrogen Ni-NTA Agarose column, taking the advantage of the histidine tag added to the protein. After the purification, enzymatic parameters would be determined by the interaction of the enzymes with the substrate; 7β-Hydroxycholesterol for cholesterol oxidase, and 5-Cholesten-3β-ol-7-one for 7-dehydratase and oxoacyl reductase.

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.