Genetic and Bacterial Manipulation

• Genetic manipulation is directly altering an organism's genes using biotechnology to achieve desired traits or functions.

Importance in Environmental Biotechnology

• Genetic manipulation is crucial for developing organisms that can degrade pollutants, improve waste management, and enhance phytoremediation.
• Genetic manipulation allows scientists to engineer microorganisms, plants, and viruses to perform specific environmental tasks efficiently.

Manipulation of Bacteria Without Genetic Engineering

• Bacteria can be manipulated without altering their genetic material by training them to adapt to specific environmental conditions or pollutants.
• Methods include improving tolerance to pollutants, enhancing degradation capabilities, and optimizing environmental conditions.

Improving Tolerance to Pollutants

• Expose bacteria to increasing levels of a pollutant (e.g., heavy metals, pesticides) in a controlled environment.
• Over time, bacteria develop mutations that allow their survival in higher concentrations of the pollutant.
• Bacteria undergo natural selection.
• Only those with mutations conferring resistance survive and reproduce.
• E. coli adapted to tolerate high levels of mercury are useful for cleaning up mercury-contaminated sites.

Enhancing Degradation Capabilities

• Culture bacteria in a medium where the pollutant is the only available carbon source.
• This forces the bacteria to evolve the ability to break down the pollutant for energy.
• Mutations occur in the bacterial genome, allowing them to produce enzymes that can metabolize the pollutant.
• Rhodococcus bacteria can degrade polychlorinated biphenyls (PCBs).
• PCBs are persistent organic pollutants.

Optimization of Environmental Conditions

• Control environmental factors such as temperature, pH, and nutrient availability to optimize the activity of bacteria for specific tasks.
• Bacteria perform better under certain conditions.
• Fine-tuning conditions enhances their natural functions like pollutant degradation.
• In wastewater treatment, bacteria are grown in aerobic conditions to enhance their ability to break down organic matter.

Acclimatization

• Acclimatization involves using naturally occurring bacteria in their unmodified form to adapt to polluted environments.
• In nature, bacteria exchange genetic material through processes like conjugation to acquire new traits, such as the ability to degrade pollutants.
• Indigenous bacteria develop the ability to degrade hydrocarbons through natural genetic exchange in oil-contaminated environments.

Manipulation of Bacteria With Genetic Engineering

• Genetic engineering involves directly altering the DNA of bacteria to give them new capabilities, such as the ability to degrade specific pollutants or produce useful enzymes.
• Methods include using plasmids as vectors, transformation, transposons, and bacteriophages.
• Genetically engineered Pseudomonas bacteria have been used to clean up oil spills by introducing genes that enhance their ability to degrade hydrocarbons.

Plasmids as Vectors

• Plasmids are small, circular DNA molecules that can replicate independently of the bacterial chromosome.
• They often carry genes for useful traits, such as antibiotic resistance or pollutant degradation.
• Scientists can insert a gene of interest into a plasmid and then introduce the plasmid into bacteria.
• The bacteria will then express the new gene.
• The nah plasmid in Pseudomonas bacteria carries genes for the degradation of naphthalene, a common pollutant.

Transformation

• Transformation occurs when bacteria take up free-floating DNA from their environment and incorporate it into their genome.
• Bacteria become "competent" under certain conditions, allowing them to take up DNA.
• Transformation can be enhanced in the lab using techniques like electroporation, where an electrical pulse makes the bacterial cell membrane more permeable.
• Escherichia coli transformed with a pET plasmid can express enzymes that detoxify heavy metals.

Transposons

• Transposons are mobile genetic elements that can "jump" within the genome, sometimes carrying new genes with them.
• Transposons can cut themselves out of one part of the genome and reinsert themselves elsewhere, potentially disrupting or activating genes.
• The Tn5 transposon has been used to introduce antibiotic resistance genes into bacteria.

Bacteriophages

• Bacteriophages are viruses that infect bacteria and can be used to transfer genes between bacterial species.
• When a bacteriophage infects a bacterium, it can integrate its DNA into the bacterial genome, potentially transferring new genes.
• Bacteriophages have been used to transfer genes for antibiotic resistance between E. coli and Salmonella.
• Phage-mediated delivery of mercury reductase genes enables bacteria to detoxify mercury in polluted water.

Restriction Enzymes and DNA Ligase

• Restriction enzymes cut DNA at specific sequences, while DNA ligase joins DNA fragments together.
• These enzymes are essential for creating recombinant DNA, where a gene of interest is inserted into a plasmid or other vector.
• The restriction enzyme EcoRI cuts DNA at a specific sequence.
• This allows scientists to insert new genes into plasmids.

Polymerase Chain Reaction (PCR)

• PCR is a technique used to amplify a specific piece of DNA, making millions of copies in a short time.
• PCR involves cycles of heating and cooling to denature DNA, anneal primers, and extend new DNA strands using a heat-stable DNA polymerase.
• PCR is used to amplify genes for insertion into plasmids or other vectors.

Gene Expression in Bacteria

• Genes coding for degradative enzymes are often arranged in clusters called operons, which are frequently located on plasmids.
• Transferring operons on plasmids allows bacteria to rapidly adapt to new carbon sources by expressing the necessary enzymes.
• The lac operon in E. coli allows the bacteria to metabolize lactose when glucose is unavailable.

Recombinant Bacteria

• Recombinant bacteria are created by introducing foreign DNA into the bacterial genome, often using plasmids or other vectors.
• Foreign DNA is inserted into a cloning vector, which is then transferred into the bacterial cell.
• The bacteria will then express the new gene.
• Recombinant E. coli have been engineered to produce insulin for medical use.

Mutagenesis

• Mutagenesis involves increasing the rate of mutation in an organism, often by exposing it to mutagens like UV radiation or chemicals.
• Mutations can lead to new traits, such as the ability to degrade specific pollutants.
• Mutagenesis has been used to create bacteria that can degrade chlorinated solvents like trichloroethylene.

Manipulation of Yeast

• Yeast, as a unicellular eukaryote, is used in biotechnology for expressing eukaryotic genes.
• Methods include cell wall removal, plasmid vectors, and integration into the host genome.
• Yeast has been engineered to produce biofuels like ethanol.

Cell Wall Removal in Yeast

• The yeast cell wall is removed using enzymes like lyticase, creating protoplasts that are more permeable to DNA.
• Once the cell wall is removed, DNA can be introduced into the yeast cell using techniques like electroporation.
• Saccharomyces cerevisiae (baker's yeast) is used in genetic engineering experiments.

Plasmid Vectors in Yeast

• Yeast plasmid vectors are designed to replicate in both bacteria and yeast.
• This allows for initial manipulations in bacterial cells before transfer to yeast.
• The plasmid contains a yeast origin of replication and selectable markers for identifying transformed yeast cells.
• The pYES2 plasmid is used for expressing genes in yeast.

Integration into Host Genome

• Yeast plasmid vectors can integrate into the host genome via homologous recombination.
• The plasmid contains sequences homologous to the yeast genome, allowing it to integrate at specific locations.
• Integration of the URA3 gene into the yeast genome allows for selection of transformed cells.

Manipulation of Viruses

• Viruses can be genetically manipulated to act as vectors for delivering genes into cells, producing proteins, or acting as biological control agents.
• Methods include recombination, cloning, and packaging.
• Baculoviruses have been engineered to produce vaccines and insecticides.

Recombination in Viruses

• Recombination is used to integrate foreign DNA into the viral genome, by replacing a viral gene with a gene of interest.
• Foreign DNA is inserted into the viral genome using homologous recombination.
• Baculoviruses have been engineered to produce human proteins for medical use.

Cloning in Viruses

• DNA manipulations are often performed on plasmids in E. coli before being transferred to the viral genome.
• The plasmid contains the gene of interest and sequences that allow it to integrate into the viral genome.
• The pFastBac plasmid is used for cloning genes into baculoviruses.

Packaging in Viruses

• Recombinant DNA is packaged into viral particles, which can then infect target cells and deliver the DNA.
• The viral genome is replicated and packaged into capsids, which are then released from the host cell.
• Adenoviruses have been used to deliver genes for gene therapy.

Manipulation of Plants

• Genetic manipulation in plants is used to improve crop yields, nutritional value, and phytoremediation capabilities.
• Methods include overcoming cell wall barriers, Ti plasmid of Agrobacterium tumefaciens, biolistic transformation, and protoplast fusion.
• Transgenic corn has been engineered to resist pests and herbicides.

Overcoming Cell Wall Barriers

• Plant cells have rigid cell walls that must be breached to introduce foreign DNA.
• Techniques like enzymatic digestion or physical methods (e.g., gene guns) are used to create openings in the cell wall.
• Enzymes like cellulase and pectinase are used to remove the cell wall and create protoplasts.

Ti Plasmid of Agrobacterium tumefaciens

• The Ti plasmid is used to transfer DNA into plant cells, causing crown gall disease.
• Scientists have modified the Ti plasmid to carry desired genes into plants.
• The T-DNA region of the Ti plasmid is transferred into the plant genome, where it can be expressed.
• The Ti plasmid has been used to create herbicide-resistant crops.

Biolistic Transformation

• DNA-coated particles are fired into plant tissue using a gene gun, allowing the DNA to enter the cells.
• The particles penetrate the cell wall and deliver the DNA into the nucleus, where it can integrate into the genome.
• Biolistic transformation has been used to create transgenic sugarcane and poplar trees.

Protoplast Fusion

• The cell wall is removed to create protoplasts, which are then fused together to combine their genetic material.
• Protoplasts are made permeable to DNA, allowing for the introduction of new genes.
• Protoplast fusion has been used to create hybrid plants with desirable traits.

Genetic Manipulation in Environmental Biotechnology

• Genetic manipulation has great potential in environmental biotechnology.
• There are challenges related to cost, containment, and competition with indigenous species.

Challenges

• Genetic engineering is expensive, and the high cost may not be sustainable for environmental applications.
• Genetically modified organisms (GMOs) must be contained to prevent unintended environmental impacts.
• Engineered organisms may struggle to compete with indigenous species in natural environments.
• Recombinant bacteria designed to degrade pollutants may be outcompeted by native bacteria in the field.

Ethical Considerations

• Genetic manipulation can have unintended consequences, such as the spread of engineered genes to wild populations.
• Strict regulations are needed to ensure the safe use of GMOs in the environment.
• Public acceptance of GMOs is a major factor in their use.
• The release of genetically modified mosquitoes to control disease has raised ethical concerns.