All the Biobricks obtained by PCR and their characterization have proved their functionality in order to be further applied for the construction of the 3 enzymes cassette and its action over 7-ketocholesterol. As it has been shown in results the promoter and BGHPA parts work as intended in mammalian cells. The suffix and prefix added through the primers design have given an advantage for an easier assembly of expression cassettes because working with standardized DNA fragments ensure in almost every case a directional insertion and junction of them. The contribution of these parts can be useful for further eukaryotic studies and constructions not only for medical applications as in this project but also investigation in other areas.

Despite the presence and expression of the proteins was proved, we couldn’t make the enzymatic analysis with their respective substrate due to lack of time. The next steps to be done after the induction of each protein with IPTG using a bacterial expression vector, would be the purification of each enzyme by affinity chromatography in a column of Ni-NTA agarose and the kinetics analysis with 7β-hydroxycholesterol for cholesterol oxidase and 5-cholesten-3β-ol-7one for 7-dehydratase and oxoacyl reductase. Our construct is designed to be expressed in mammalian cells, specifically macrophages, to help the human body metabolize oxidized cholesterol molecules, such as 7-ketocholesterol. With a correctly assembled vector made from all the biocricks, it will be possible to design of a new alternative treatment to prevent the formation of the atherosclerotic plaque. Considering the local and international normative, we are tending to apply this treatment to people with potential risk to develop this condition.


According to the results obtained we are able to conclude that the approach taken for the development of the project was appropriate: recombinant protein was obtained and all of the biobicks were properly assembled. According to the differential model obtained, 7-ketocholesterol metabolism should be observed when performing the enzymatic assay; however, due to a lack of time we were unable to perform it and present further results. On the other hand, we were able to generate the following biobricks: BGHPA (polyadenylation signal), CMV (eukaryotic promoter), NeoR (eukaryotic selective marker), which had not been reported before at iGEM registry and will allow teams to work with mammalian cells in the close future. Coding sequences for oxoacyl reductase and 7-dehydratase were also assembled following iGEM’s guidelines.

Perspectives

Given the obtained results, future iGEM teams can be able to follow up our project and continue to achieve objectives such as: construction of a plasmid containing all of the coding sequences generated by our team as follows: CMV promoter + RBS + oxoayl reductase + 7-dehydratase + cholesterol oxidase + BGHPA with NeoR as a selective marker for eukaryotic cells; transfection of a macrophage cell line, and enzymatic assay and characterization.