Microbial Bioengineering and CRISPR Applications

Introduction to Microbial Bioengineering

• Microorganisms can be engineered to metabolize raw materials from nature for producing various compounds, including polymers.
• Benefits include environmentally friendly production methods and potential for applications in medication, agriculture, biosensing, and scientific research.

Applications of Engineered Microorganisms

• Production of Polymers: Microorganisms can be modified to produce biodegradable polymers instead of conventional plastic, leading to reduced environmental impact.

• Antibiotics Mass Production: Microorganisms can be engineered to synthesize antibiotics in bulk, meeting medical demands effectively.

• Agricultural Enhancements: Engineered microorganisms can improve nutrient absorption by plants, facilitating healthier growth.

• Biosensing: Specialized microorganisms can detect specific compounds in environmental samples, like water or soil, using fluorescence as an indication of presence.

  • Example: A microorganism that fluoresces green or red upon detecting certain pollutants.

Bioengineering Tools

• Development of new tools is vital for creating complex microbial systems and studying unknown biological mechanisms.
  • Synthetic Cell Project: This initiative aims to construct a functional cell from scratch, investigating the minimal genetic requirements for life.

Plasmid Assembly Approaches

• Traditional plasmid construction methods can be lengthy, taking months. Efficient approaches are required for bioengineering.
  • CYADRUM coding and Gibson Assembly: These methods utilize altered gene sequences that act as building blocks (like LEGO pieces) to streamline plasmid construction, allowing for rapid assembly of multiple plasmids (20-30 in a month).

Gene Regulation Through Recombinases

• Recombinases: Proteins that mediate DNA recombination processes, important for adapting genetic information.
  • Two common mechanisms include:
  1. Deletion Integration: Cuts and relocates specific DNA segments in response to bacteriophage infections.
  2. Inversion: Flips sections of DNA to alter gene expression without permanent deleting sequences.

CRISPR Systems Overview

• CRISPR Components: Key components include guide RNA, CRISPR RNA, and signature Cas9 protein for targeting and editing DNA sequences.
• CRISPR Activation (CRISPRa): Utilizes a deactivated Cas9 (dCas9) to bind DNA without cutting it, enabling regulation of gene expressions through interaction with transcription factors.
• SOX s Transcription Factor: Requires cofactors for binding its target, which can be facilitated by using a CRISPRa complex.

Inducible Gene Expression Systems

• The project aims to establish a system that can control gene expression in a bacterial cell and induce behaviors such as switching between producing different fluorescent proteins (GFP and RFP).
• Cell Memory: The goal is to give bacterial cells the ability to remember and switch between states based on induced commands, akin to memory in computers.

Experimental Workflow

• Steps executed include:
  1. Plasmid construction and transformation into bacterial cells.
  2. Culturing on antibiotic plates for selection of successful transformations.
  3. Incubation and analysis using plate readers to measure expression levels of fluorescent proteins.

Results Interpretation

• Successful transitions between behaviors (like switching from RFP to GFP) indicate the correct functioning of the system.
• Future experiments will test the system's responses to different concentration levels of inducers like arabinose to eliminate issues like leaky expression.

Future Directions

• Aim to enhance CRISPR activation systems to allow for additional behavioral switches, potentially enabling three states (GFP, RFP, and another color).

• Further development in the recombination origin project seeks to refine plasmid replication control.

  • Testing various origins of replication for improved efficacy and stability in expression

• Explore solutions for observed leak and strain issues, including refined ribosome binding sites and regulatory sequences.

Conclusion

• The research aims to create sophisticated microbial systems capable of responding to environmental stimuli, which has applications in disease detection, agriculture, and sustainable manufacturing.
• Continued improvements in bioengineering tools are essential to realize these applications and enhance knowledge of biological systems.