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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster H20 - Orthogonally controlling the expression of the shell protein and assembly factors enables optimally controlled assembly of gas-filled protein nanostructures in heterologous hosts

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Gas vesicles (GVs) are protein nanostructures that convey buoyancy to microbial cells. Their assembly inside cells requires an orchestra of the shell protein and approximately 10 assembly factor proteins. Since the assembly factors act as enzymes to catalyze the assembly of the shell proteins, the assembly factors likely exist in much lower copy numbers and do not get consumed during the assembly process. Previous experimental data support the existence of a much higher transcriptional rate of the shell protein compared to the assembly factors. While the stoichiometry between the assembly factors and the shell protein is tightly regulated in the native host cells, such regulation is usually lost when expressed as transgenes in heterologous hosts. In the recent applications of GVs as acoustic reporter genes in therapeutic microbes and mammalian cells, a simple single-inducer system was used to drive the expression of both the shell and assembly factor proteins. As a result, a large cell-to-cell deviation in the amount of successfully assembled GVs was observed within a given cell population, hinting at cytotoxicity and mutations. Here, we hypothesize that a system orthogonally controlling the expression of the shell protein and the assembly factors can approximate the optimal stoichiometry for the assembly of GVs in heterologous hosts.

Materials and

Methods:  First, we designed a genetic circuit to orthogonally control the expression of the shell protein and assembly factors. The gene operon derives from Priestia Megaterium, in which its GV gene cluster encodes the main shell protein, GvpA2, and other 10 assembly factor proteins. The operon is split into two parts controlled by two orthogonal chemical inducers. We then validated the performance of this plasmid design by testing it with fluorescent proteins.
We also found that optimizing the dual-inducer system could eliminate the drop in optical density. Previous plasmid design led to a sharp drop of E. coli culture optical density, indicating cytotoxicity. The newly designed genetic circuit was transformed and expressed, then induced with two chemical inducers. One of the inducers was tested with varying concentrations to test the effect of different concentrations on the expression of GVs. The optical density was tracked every hour at a wavelength of 600 nm. Hydrostatic collapse testing was performed to quantify the amount of GVs.

Results, Conclusions, and Discussions: The previous E. coli expression design had a drop of its optical density post-induction, indicating cytotoxicity. Our new design of the circuit was able to eliminate such a drop. Testing the GVs induced with various concentrations of inducer under hydrostatic pressure showed a decreasing trend of the percent drop in optical density as its concentration increased. Our results represent an important step towards creating an optimal expression system for using GVs as multimodal reporter genes and also serve as an important example of understanding the regulation and assembly process of protein organelles.

Acknowledgements (Optional):