CRISPRi engineering E. coli for morphology diversification.

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Bacterial cell shape is determined by genes ftsZ and mreB, crucial for cell division and structure.
• Microbial morphology engineering is a growing field with significant biotechnological potential.
• Controlling gene expression is key to manipulating bacterial cell morphology.

Purpose of the Study:

• To investigate the use of CRISPR interference (CRISPRi) for precise regulation of ftsZ and mreB gene expression in E. coli.
• To engineer E. coli cell morphology by modulating the expression of ftsZ and mreB.
• To explore the relationship between altered cell morphology and intracellular polyhydroxybutyrate (PHB) accumulation.

Main Methods:

• Designed and synthesized five single-guide RNAs (sgRNAs) targeting different locations on ftsZ and mreB genes.
• Utilized CRISPRi technology to achieve varying degrees of gene expression repression.
• Combined multiple sgRNAs to intensify gene expression repression.
• Quantified changes in cell length, width, morphology, and intracellular PHB content.

Main Results:

• CRISPRi successfully reduced ftsZ and/or mreB expression, leading to elongated and/or fatter E. coli cells.
• Stronger repression correlated with increased cell length and volume.
• Combined sgRNA repressions produced diverse cell morphologies (gourds, bars, cocci, spindles, etc.).
• Intracellular PHB accumulation was directly proportional to increased cell volume, ranging from 40% to 80%.

Conclusions:

• CRISPRi is an effective tool for microbial morphology engineering by precisely controlling bacterial cell shape.
• Modulating ftsZ and mreB expression via CRISPRi offers a method to generate E. coli with diverse and enlarged morphologies.
• Increased cell volume resulting from CRISPRi-mediated gene repression leads to proportional accumulation of PHB, suggesting potential for biopolymer production.