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High School Poster

HS Poster

Poster 95 - Investigating the Function of Arabidopsis Thaliana Transcription Factor BBX31 in Saccharomyces Cerevisiae​

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

    Presenting Author(s)

  • Zixu Chen (he/him/his)

    Intern
    University of California San Diego: Institute of Engineering in Medicine
    San Diego, United States

    • Co-Author(s)

  • Yangyi Liu

    Undergraduate researcher
    UCSD, United States

  • CF

    Conner Feng

    freshman undergraduate, mentor
    University of California San Diego: Institute of Engineering in Medicine, United States

    • Co-Author(s)

  • Zechun Wang

    intern
    University of California San Diego: Institute of Engineering in Medicine
    San Diego, California, United States

  • Alan Zhang

    Intern
    University of California San Diego: Institute of Engineering in Medicine, United States

    • Co-Author(s)

  • EC

    Ethan Chin

    intern
    University of California San Diego: Institute of Engineering in Medicine, United States

  • KW

    Kaibiao Wang

    Postdoc
    University of California San Diego: Department of NanoEngineering, United States

  • VG

    Veronica Gomez-Godinez

    Scientist
    University of California, San Diego
    La Jolla, California, United States

  • Linda Shi, PhD (she/her/hers)

    OPALS Program Director, Project Scientist, Co-director IEM
    University of California, San Diego: Institute of Engineering in Medicine
    La Jolla, California, United States

    • Primary Investigator(s)

  • YL

    Yanran Li

    Principal Investigator
    University of California San Diego: Department of NanoEngineering, United States


Introduction: Engineered Saccharomyces cerevisiae is an ideal platform for the reconstitution of various biosynthetic pathways, including the phytohormones carvacrol and gibberellin. B-box domain proteins (BBX) proteins are transcription factor proteins that are commonly found in plants, playing an essential role in controlling gene expression. Campesterol is a major phytosterol that plays an important role in the regulation of plant cell membrane properties. This study investigated how the growth of yeast cells was affected after the transcription factor BBX31 in Arabidopsis thaliana was introduced into campesterol-producing yeast. Based on protein structure analysis and experimental phenomena, a hypothesis of the connection between BBX31, campesterol, and engineered yeast growth was formulated. This study would provide potential support for utilizing exogenous transcription factors to regulate yeast gene expression and establish efficient biosynthetic pathways to obtain desired metabolites for industrial or pharmaceutical needs.

Materials and

Methods: For bioinformatic analysis, sequences of BBX family proteins were curated using Phytozome database and analyzed using Molecular Evolutionary Genetics Analysis (MEGA) software. The sequence alignment was aligned by Clustal Omega and visualized by ESPript 3.0. The proteins’ various physical and chemical parameters were computed using Expert Protein Analysis System (ExPASy) from the Swiss Institute of Bioinformatics. The structures of BBX proteins were predicted by AlphaFold Protein Structure Database and compared using Python Molecular Graphics (PyMol) software. Yeast strains were cultured at 30 °C in synthetic defined medium (SDM). Cell size under various conditions was monitored with Zeiss Axiovert 200M microscope. Optical density at 600 nm was measured with Molecular Devices NanoDrop. Spotting assays were performed on SDM agar plates and imaged with Bio-Rad gel scanner under white light setting. Image analysis was conducted with CellProfiler and Fiji ImageJ, followed by analysis of variance (ANOVA) and post hoc in R.

Results, Conclusions, and Discussions: The campesterol-producing yeast strain showed growth deficiency compared to the wild type due to the knockout of genes in ergosterol biosynthesis that are important for membrane regulation in yeast. Compared with the unmodified campesterol-producing yeast, overexpressing BBX31 significantly improved cell growth on agar plates. But in aqueous solution, the yeast growth demonstrated complexity. In 4.75% EtOH and 0.5M NaCl, the modified BBX31 yeast growth was depressed, and there was no significant difference in cell area. The cell length of modified yeast and unmodified yeast was measured under isotonic (SDM), hypotonic (distilled water), and hypertonic (0.5M NaCl) conditions. The cell diameter of BBX31 modified yeast decreased significantly compared to unmodified yeast under hypertonic condition. The results of OD 600 showed that both modified yeast and unmodified yeast have a higher growth rate at the presence of 0.5M NaCl compared to those without NaCl, and the unmodified yeast even grew better in 0.5M NaCl at middle and late stage. The results demonstrated that the effects of BBX31 on campesterol-producing yeast growth are multifaceted and complex. We hypothesized that BBX31 functions as a transcription factor regulating genes involved in synthesis of membrane in yeast. By influencing the incorporation of campesterol into the cell membrane, BBX31 rescues the ergosterol deficiency while affecting the property of cell membrane, thereby leading to different growth patterns in the yeast cells.

Overexpression of BBX31 affects the osmotic response of campesterol-producing yeast, indicating BBX31 regulates genes involved in the integration of campesterol into the cell membrane. In future research, measuring the levels of different phytosterols (especially campesterol and ergosterol) in yeast and the permeability of yeast cell membranes is important. To understand the mechanism of how BBX31 regulating the gene expression of yeast, using chromatin immunoprecipitation sequencing (ChIP-Seq) to identify its binding sites is worth considering. Studying how other BBX proteins from Arabidopsis thaliana affect the campesterol-producing yeast growth will also provide support for understanding the mechanism of BBX31.

Acknowledgements (Optional): This work was supported by a generous gift from Beckman Laser Institute Inc. to V. G. G. & L. S. and the National Science Foundation under Award Number 2420331. The high school students (*) participated in the UCSD IEM OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering, Dr. Lizhu Chen from CorDx Inc. for their donations, and Dr. Shanhui Xu for providing the yeast strains.

References (Optional):