Proof of Concept

Golden Gate Cloning

Figure 1. The six transcriptional units (TU) we attempted to assemble, using our parts collection
Figure 2. Successfully assembled TU A (lane 2) and JUMP plasmid vector (3), digested by Esp3I; the transcriptional unit was submitted as the composite part BBa_K4728016.
Figure 3. JUMP plasmid vector (lane 2) and successfully assembled TU B (3), digested by Esp3I; TU B was submitted as the composite part BBa_K4728017
Figure 4. JUMP plasmid vector (pJUMP29-1C) and successfully assembled TU C1, C2, and C3, digested by Esp3I. Two minipreps of putative TU D were prepared, but neither were successful, as bands were expected at 3953, 2973, and 980bp (in yellow). TU C2 was submitted as the composite part BBa_K4728019 and C3 as the composite part BBa_K4728020.
Figure 5. Positive colonies with TU C1 express eiraCFP.

Future directions would be to confirm protein expression. Positive TU C1 colonies express the reporter eiraCFP, suggesting that the CDS upstream, tphC, is also being expressed. However, a protein extraction SDS-PAGE would provide further conclusive evidence. Another next step would be to take multiple TUs and assemble them into a level 2 cloning vector. These composite parts are meant to work together; more information can be found on the Description, Engineering, and Parts pages.

Phasin Mutant Modelling

Our homology structure captures the universal properties of phasins, featuring alternating hydrophobic and hydrophilic regions, essential for their surfactant function. Notably, it includes a conserved N-terminal domain with α-helix conformation and a leucine zipper domain housing their oligomerization capability.

Figure 6. Homology structure of phasin, created through alphafold 2

To ensure the fidelity of our homologous structure, we conducted energy minimizations considering pressure, temperature, and volume in GROMACS.

Figure 7. Demonstrating the homologous protein of PhaF was stable and contained the correct domains

We employed AutoDock Vina for our docking studies, leveraging genetic algorithms and local optimization for precise ligand-receptor interaction prediction. This provided deeper insights into the wildtype and mutant interaction. The docking results highlighted significant differences in binding affinity. The Mutant:Monomer exhibited a binding affinity of -2.8 kcal/mol, while for Wildtype:Monomer it was -2.0 kcal/mol, both at an RMSD score of 0.0.

Figure 8. PHB monomer binding to Bi1 domain of mutant, binding affinity is indicated, it interacts with LEU, GLY & TRP.

Due to time constraints, we weren't able to conduct Molecular dynamic (MD) simulations accounting for both our wildtype and mutant in relation to the PHB membrane. However, our proposed model holds promise in light of our docking results. Below we have constructed a prepared minimized CHARRM-GUI model for our potential MD.

Figure 9. Proposed model for our membrane protein interaction, built in CHARRM-GUI