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Related Concept Videos

Production and Aerosol-Based Deposition of Bacillus subtilis Spores03:37

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Source: Fuchs, F. M., et al. Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy. J. Vis. Exp. (2017)This video demonstrates the production, purification, and aerosol-based deposition of Bacillus subtilis spores to create a uniform monolayer, enabling downstream applications such as microscopy-based analysis of spore germination, viability, or stress...
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Related Experiment Video

Updated: Jan 20, 2026

Production and Aerosol-Based Deposition of Bacillus subtilis Spores
03:37

Production and Aerosol-Based Deposition of Bacillus subtilis Spores

Published on: October 30, 2025

294

Controlling Heterogeneity and Increasing Titer from Riboswitch-Regulated Bacillus subtilis Spores for Time-Delayed

Denis Tamiev1, Alyssa Lantz1, Grace Vezeau2

  • 1Department of Biochemistry Biophysics and Molecular Biology , Iowa State University , Ames , Iowa 50011 , United States.

ACS Synthetic Biology
|September 7, 2019
PubMed
Summary

Sporulated cells can be engineered for on-demand protein production, but maintaining consistent expression rates after germination is challenging. This study investigates plasmid types and bacterial mutants to improve protein expression consistency from spores.

Keywords:
delayed protein productionfield-usespacesporessynthetic biology

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Last Updated: Jan 20, 2026

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Area of Science:

  • Synthetic Biology
  • Microbial Engineering
  • Protein Expression Systems

Background:

  • Sporulated bacterial cells offer potential as controllable protein expression platforms for applications like biologics and vaccines.
  • Achieving high and consistent protein expression rates from germinated spores remains a significant challenge.
  • Variability in expression post-sporulation can hinder the reliability of these engineered microbial systems.

Purpose of the Study:

  • To investigate the impact of integrating versus theta-replicating plasmids on protein expression from *Bacillus subtilis* spores.
  • To evaluate the role of PolY mutants in mitigating expression variance after sporulation.
  • To quantify the effects of different genetic constructs and mutations on protein expression kinetics and variability.

Main Methods:

  • Engineered *Bacillus subtilis* strains (wild-type and PolY mutants) to express a theophylline-inducible fluorescent reporter protein (RFP).
  • Utilized integrating and theta-replicating plasmids to assess differences in expression cassette copy number.
  • Analyzed fluorescence and cell-growth curves using a custom kinetic model to determine peak kinetic rates (LKPmax) and variances.

Main Results:

  • Plasmid-based expression resulted in 8.7-fold higher expression rates compared to integrated constructs due to increased copy numbers.
  • The variance of expression rates (LKPmax) increased 2.1-fold after sporulation in the wild-type strain.
  • PolY knockouts partially mitigated the increase in expression variance in both suspended cells and biofilms.

Conclusions:

  • Theta-replicating plasmids enhance protein expression rates from *Bacillus subtilis* spores but can increase expression variance post-sporulation.
  • The increased variance post-sporulation is comparable to UV exposure effects and can be partially overcome using PolY mutants.
  • Optimizing plasmid choice and bacterial strain genetics is crucial for reliable, high-yield protein production from sporulated cells.