Poster X4 - Enhanced Visualization of Gas Vesicles by Expressing Reporter Gene Using Gas Vesicle Protein C

Introduction: Gas vesicles (GV) are air-filled proteins that self-assemble inside bacteria to help them float. These nanostructures scatter sound waves for ultrasound imaging. Recently, GVs were developed as imaging and delivery contrast agents [1]. Gas vesicle protein A (GvpA) and C (GvpC) are main structural proteins that provide mechanical integrity to GVs. As GvpC mainly contributes to the formation of outer shells of GVs, we used GvpC to express foreign proteins by making a fusion between them. As the fusion between GvpC and foreign protein affects the formation of GVs and their expression, we tested the expression of both reporter gene and GVs by inserting mNeonGreen (mNG) fluorescent proteins at N-terminus of gas vesicle protein C genes (gvpC).

Materials and

Methods: A plasmid encoding gvpA from Anabaena flos-aquae (Ana) and accessory genes gvpR–gvpU from Bacillus megaterium (Mega) was obtained from Addgene (ARG1, catalog number #106473). mNG sequence was PCR amplified using Q5 DNA polymerase and forward and reverse primers. ARG1 was digested using CutSmart at SacII and NheI sites. The PCR product of mNG and digested ARG1 were ligated using T4 DNA Ligase to construct mNG-gvpC (mNG-ARG1). The plasmids were then transformed in One Shot BL21-Al chemically competent cells, which were then cultured in super optimal broth with catabolite repression (SOC) outgrowth media with appropriate antibiotic for 16 hours at 37 °C. Then a large culture was started with 1ml of the small culture with 100 ml of LB media, kanamycin, and glucose. BL21 were grown at 37 °C until optical density at 600 nm (OD600) reached 0.5. Cells were induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 22 hr. at 30 °C. Cells were collected and pelleted by centrifugation at 350 × g in 50 ml conical tubes at 25 °C for 4 hr with a liquid depth ≤ 10 cm. Buoyant part was separated and imaged using phase contrast microscopy and epi-fluorescence microscopy. All images were taken using 100X objective lens.

Results, Conclusions, and Discussions: Result &

Discussion: mNG and GVs were simultaneously expressed in BL21 after mNG-ARG1 were transformed (Fig. 1A). Based on the number of cells expressing mNG and GV, no interference between two proteins was observed. For verification of the result, a positive control (ARG1 without mNG, Fig 1B) and a negative control (BL21 without ARG1 nor mNG-ARG1, Fig 1C) was tested. They showed neither mNG nor GV expressions.

Conclusions: We can use mNG expression to locate the position of GVs and BL21. Our work shows that the insertion of mNG on the N-terminus of GvpC expresses does not interfere with each other, which indicates that other complex functional proteins may be fused with gvpC for other purposes. While previous research has extensively focused on the foreign protein insertion at the C-terminus of GvpC, our results demonstrate that N-terminus can be used for foreign protein fusion. Given the potential of GvpC for bioengineering GV and displaying foreign protein sequences on the surface of the gas vesicles, our work expands the scope of hybrid expression with other foreign proteins for the development of gene therapy and therapeutic delivery with these unique air-filled nanostructures.

Acknowledgements (Optional): This work is partially supported by the National Institute of Health (NIH) grant No. GM152704 and the National Science Foundation (NSF) grant No. CBET1943852.

References: [1] Kim, Sangnam, Siyuan Zhang, and Sangpil Yoon. “Multiplexed Ultrasound Imaging Using Spectral Analysis on Gas Vesicles.” Advanced healthcare materials. 202200568 (2022)