Overview

Engineering plays a crucial role in synthetic biology research. Similar to other engineering disciplines, developing a biological system is a multifaceted, demanding, and extensive endeavor that can sometimes progress at a slow pace. This process follows an engineering cycle with four main stages: design, build, test, and learn. In our project, we repeat this cycle continuously. The steady accumulation of engineering outcomes ultimately brought us to the project's final objective.

In the search to develop a gene expression mechanism dependent on the presence of EE2, we highlight three engineering cycles: protein-EE2 affinity (First engineering cycle), recognition of protein DNA binding domain (Second engineering cycle) and experimental validation of protein function (Third engineering cycle).

1st iteration

Protein-EE2 affinity

Design

Reading in literature we found a TetR-like protein with affinity for estrogens, especially estrogen (E2). However, its affinity for EE2 was minimal, so we used this protein as a starting point to modify its allosteric site, not only allowing the coupling of EE2 but also increasing its affinity.

Build

We mutate the allosteric site at each iteration by rationally modifying amino acids in FASTA and designing new proteins in AlphaFold 3.

Test

To test the performance of our mutations, we ran semi-flexible dockings with Autodock Vina and refined them by conducting flexible molecular docking with Rosetta Ligand-Docking.

Learn

After the previous steps, we observed the Rosetta Scores and decided whether to keep this mutation; if we kept it, we proceeded to determine whether it was necessary to make a new mutation on it or not.

This process was repeated until we had a Rosetta Score higher than that of the protein with E2.

2nd iteration

DNA binding site

Design

In the previous cycle we were able to make our protein EE2-like, however its DNA binding site still belonged to that of the Tet-R like original protein, a binding site that is not documented and unknown. For this reason, we modified the binding site of our protein in order to have a recognized binding domain and thus implement it in genetic circuits.

Build

The protein mutation of the binding site was done by modifying the FASTA sequence, removing the original sequence and adding the recognized sequence of the original documented Tetracycline protein.

Test

For the in silco validation we ran molecular dynamics simulations between different molecules involved in this interaction to model the evolution of the system in time.

Learn

After each test we decided whether to make any modifications to the extra binding site for greater affinity or practicality, this was repeated until we had a recognized and stable sequence that did not alter our previously achieved allosteric modification.

3rd iteration

Proof of concept

Design

In order to test in the laboratory the correct functioning of what was achieved in silico, a plasmid containing our designed protein ESTER and a MCherry fluorescence macerator was constructed, with the intention of proving that our culture (E.coli BL21) transformed and induced represses gene expression when there is no EE2 in the medium and allows gene expression in the presence of EE2.

Build

The construction of this experiment was carried out by inoculating our transformed cells in a 50 ml culture, this inoculum was monitored until it reached an Optical Density of 0.6 (at 600 nm), when it reached this growth it was induced with IPTG, incubating a control group without EE2 and an experimental group with EE2.

Test

To test our hypothesis, after lysing and quantifying each group, the fluorescence yielded by each was measured; we expected to obtain a much higher fluorescence in the experimental group and a lower one in the control, parameterizing the measurements with the MCherry fluorescence values.

On the other hand the simulations were analyzed to evaluate the biological activity of the protein.

Learn

Following our initial test, we evaluated the accuracy of our results and, based on this assessment, implemented experimental modifications for subsequent trials or conducted additional replications to achieve more precise and reliable outcomes.