Difference between revisions of "Part:BBa K3809010"

Biology and characterization of BBa_K3809010

We wanted to test if our protein receptor would fold adequately. However, before we could enter the lab, we could not do it experimentally, so we recurred to an in silico analysis. We wanted to verify that all the modifications and features we added wouldn’t interfere with the correct folding of BBa_K3809010; furthermore, we wanted to verify that the His Tag we added and the Linker sequence would be exposed enough for us to perform an efficient purification and an adequate immobilization. We tested this through the prediction of a protein model made using the Software Robetta [1]. After we took the best model Robetta could offer, we performed a Structural Alignment using the Human Estrogen Receptor Alpha 2IOG from PDB [2] as reference. Our results confirmed that the folding of the protein wasn’t being affected at all.

Figure 1. Protein designed by iGEM TecCEM. It includes the whole receptor hER⍺ (red), a linker sequence (green), a His Tag (magenta) and a Signal Peptide Sequence (blue). It can be seen that both the linker sequence and the His Tag are exposed. Protein visualized with Chimera [3].

Figure 2. Structural alignment between the protein designed by iGEM TecCEM (red, magenta, blue and green) and the crystallized structure of the Human Estrogen Receptor Alpha 2IOG (white). The alignment shows a preserved structure in the Ligand Binding Domain of the modified ESR1. Protein visualized with Chimera [3].

We also performed a Molecular Docking to verify its affinity towards a group of common EDCs: Estradiol (the natural ligand), Benzene, Bisphenol A, Dimethyl Phthalate, Ethylene Glycol, Phenol, Chloroprene and Perfluorooctanoic Acid. To prepare the model for the analysis, we added hydrogens to it, improving the quality of the assay. To this end, we used the tool MolProbity [4]. The ligands were obtained from PubChem [5] and the Docking was conducted using the Software tool SwissDock [6]. From the best result of the docking, we obtained its Free Gibbs Energy and used it to calculate the Association Constant. We also analyzed the type of interaction. Furthermore, we evaluated the formation of hydrogen bonds between the receptor and the small molecule. The results are seen below.

Table I. Results obtained from the Docking Analysis. Estradiol (highlighted) is the natural ligand of the receptor.

To characterize the DNA fragment of ESR1 we inserted our gene in pSB1C3 and transformed it in E. coli DH5α. To verify that the insert was cloned correctly, we extracted the plasmid from a DH5α culture and performed a PCR using M13 primers. We got positive results shown below.

Figure 3. Amplification of the ESR1 fragment to verify a correct cloning in a pSB1C3 vector.