Microbes as Tools

• • Bacteria are increasingly utilized in industrial processes as "cell factories" to produce specialty products like detergents, plastics, fuels, and break down environmental contaminants such as pesticides, petroleum additives, and plasticizers.
• • Biomineralizing bacteria are now being employed in self-healing construction materials.
  • Despite typically being associated with damage and decay in the built environment, bacteria can act as bioengineers.
  • They contribute to natural processes like sediment formation in marine and freshwater environments, and stalactite/stalagmite formation in caves.
• • Bacteria promote the precipitation of mineral carbonate, like calcium carbonate, through metabolic processes, increasing pH and carbonate concentration.
  • This precipitation fills pore spaces in soil, acting as a binding agent and leading to densification.
  • Bacterial cell surfaces, typically negatively charged, attract positively charged calcium ions, aiding in calcite crystal formation.
  • The success of using microbes in soil improvement has prompted exploration of their potential in the construction industry.
• • In geotechnical applications, bacteria consolidate and stabilize soil through carbonate mineral precipitation.
• • Bacillus and related bacteria are suitable for harsh concrete environments and can form spores for long-term survival.
  • Inclusion of bacteria in concrete allows for growth and precipitation of calcite minerals, sealing cracks.
• • Methods to optimize bacteria survival in concrete are being developed, including encapsulation in porous solids like perlite or polymer-based microcapsules.
  • Research aims to enable bacteria to perform multiple healing cycles by germinating from spores, inducing calcite precipitation, and returning to spore form.
  • Upscaling from lab to commercial scale remains a challenge.
  • Self-healing concrete technology is valuable for difficult-to-access structures like tunnels and bridges.
• • Microbial activity could have both positive (e.g., reducing gas pressure) and negative (e.g., accelerating corrosion) impacts on repository integrity.
  • Microorganisms may affect the performance of repository barriers like swelling clays, potentially reducing their effectiveness.
  • Some microorganisms can reduce the mobility of radionuclides like uranium, potentially aiding in waste containment.
• • Smart precision bio-insecticides, using bacteria, offer environmentally friendly alternatives for pest control and disease prevention.
• • Viral vectors efficiently deliver therapeutic genes, bypassing the host immune system.
• Antibiotics:

  • Fungi or bacteria
  • Penicillin
  • Streptomycin- Streptomyces griseus
  • Vancomycin-Amycolatopsis orientalis
• Sewage -> organic acids -> CH3COOH, H2, CO2 ->Methanogens -> CH4, H2O, CO2
• The TOL operon is a cluster of genes located on the TOL plasmid that encode enzymes responsible for the degradation of aromatic compounds. These enzymes act sequentially to break down molecules like toluene into intermediates that can be further metabolized by the Pseudomonas putida for energy and carbon.
• Pseudomonas putida strains carrying the TOL plasmid are often found in environments contaminated with aromatic compounds, such as soil or water polluted by petroleum products. The ability of these bacteria to degrade such pollutants is of significant interest for bioremediation efforts, as they can help break down harmful chemicals and reduce environmental contamination.
• Psdeudomonas TOL plasmid (pWW0): degrades toulene and xylenes to acetaldehyde and pyruvate
• Microbial enzymes:

  regioselective- discrimination between similar parts of the same molecule

  stereoselective- act on or generate single optical isomers
• Commercial enzyme sources:

  • Animals: rennet, trypsin
  • Plants: amylase, Beta-glucanase, papain
  • Bacteria (mainly bacillus): amylase, subtilisin, glucose isomerase
  • Yeast (saccharomyces): amylase, glucoamylase, pectinase
  • Fungi (aspergillus): invertase, lactase, lipase, raffinase
• Heterologous Expression: This involves expressing a gene in a host organism that is different from the organism where the gene naturally occurs.
• 1. Homologous Expression: This refers to expressing a gene in a host organism where the gene naturally occurs.
• Expression Plasmids: These are circular DNA molecules used to introduce foreign genes into host cells for protein expression. They often contain a promoter region for gene transcription, as well as other regulatory elements.

  • Integrative Plasmids: These plasmids can integrate into the host cell's genome.
  • Non-integrative Plasmids: These plasmids do not integrate into the host cell's genome and typically exist as episomes (extrachromosomal elements).
• 1. Promoters: These are DNA sequences that initiate the transcription of a particular gene. Strong promoters are efficient in driving gene expression, and inducible promoters allow control over when the gene is expressed.
• Affinity Purification


  A technique used to isolate a specific protein or group of proteins based on their binding affinity to a particular ligand. This method typically involves attaching a tag to the protein of interest, which facilitates its purification.
• Poly-His Tag


  A short sequence of histidine residues (usually 6-10 His residues) that can be fused to a protein. Proteins with this tag can be purified using immobilized metal affinity chromatography (IMAC), which exploits the strong affinity between histidine residues and certain metal ions.
• Glutathione-S-Transferase (GST) Tag


  GST is a protein tag derived from the enzyme glutathione S-transferase. It is commonly used for protein purification because it binds specifically to glutathione, allowing for easy purification using glutathione affinity chromatography.
• Schistosoma japonicum GST


  This likely refers to a GST tag derived from the organism Schistosoma japonicum, which would be used in a similar manner to other GST tags for protein purification. The mention of "26kDa" indicates the size of the tagged protein.
• Expression Hosts - Escherichia coli (E. coli)


  • Simple, well-characterized genetics
  • Inexpensive and fast to grow
  • Easily manipulated, lots of available vectors
  • Lots of available strains, many already genetically modified for optimal heterologous expression
• Overexpressed protein often produced as partially folded/unfolded, inactive, insoluble inclusion bodies
• Challenges with E. coli Expression


  • Inclusion bodies formation
  • Inactive protein
  • Insolubility
• Strategies to Address Challenges


  1. Co-expression of molecular chaperones

  2. Optimization of temperature, growth medium, expression rate

  3. Inclusion body purification and refolding
• Bacteria do not possess glycosylation machinery
• Glycosylation


  The process of attaching sugar molecules to proteins, which is a crucial post-translational modification for many proteins' structure, stability, and function
• The absence of glycosylation machinery in E. coli means that proteins produced in this system will not undergo glycosylation unless additional modifications are made to the host strain
• Animal and insect cells


  • Require specialized growth media containing specific nutrients and supplements
  • More costly compared to bacterial expression systems
  • Grow more slowly
  • Reach lower densities compared to bacteria
• Adherence vs. Suspension


  Some animal or insect cells require a surface (adherence) to grow, while others can grow in suspension (free-floating)
• Adherent cells


  • May need special culture vessels or coatings to facilitate attachment
  • Can add complexity to the cultivation process
• Animal and insect cell cultures

  • More prone to contamination by bacteria, fungi, or other microorganisms compared to bacterial cultures
  • Maintaining sterility is crucial to avoid contamination, which can compromise the quality of the protein product
• Transfecting animal or insect cells

  • Challenging compared to bacterial transformation
  • Even if successful, the copy number of the introduced plasmids is often low, leading to lower levels of protein expression
• Achieving stable, long-term expression of the target protein in animal or insect cells


  • Often requires integrating the gene encoding the protein into the host cell's genome
  • Can be labor-intensive
  • May result in variable expression levels depending on the site of integration
• Animal and insect cells

  • May be more sensitive to high levels of protein expression, which can lead to cellular stress or even cell death
  • Careful optimization of expression conditions is necessary to prevent adverse effects on cell viability and protein quality
• Viral transfection


  • Can achieve high efficiency in animal or insect cells
  • Viral vectors can deliver genes into the host cells more effectively, enhancing the chances of successful protein expression
• Animal or insect cells

  • Able to perform post-translational modifications, such as glycosylation
  • Can be important for proteins that require proper folding or functional activity