week 5 and 6 at BioHack Academy Waag 2024

with TOM PEETERS and MATTHIJS DE BLOCK

Genetic modification (GM), also known as genetic engineering, is a process by which scientists alter the genetic material of an organism. This can involve adding new DNA, removing DNA, or altering an organism’s genetic makeup in a way that does not naturally occur. This technology is applied in various fields, including agriculture, medicine, and research.

In agriculture, GM is used to enhance crop resistance to pests and diseases, improve crop yields, and increase nutritional value. For example, genetically modified crops like Bt corn are engineered to produce a bacterium that is toxic to specific insect pests.

In medicine, genetic modification has led to the development of gene therapy, which aims to treat or prevent diseases by directly altering the genetic material of a person’s cells. Another significant advancement is the production of genetically engineered organisms that can produce pharmaceuticals, such as insulin and vaccines.

Despite its potential benefits, genetic modification raises ethical, environmental, and health concerns. For instance, there are debates over the safety of GM foods for human consumption and the potential for genetically modified organisms to affect biodiversity.

Regulations and public acceptance of genetic modification vary widely between countries, reflecting the diverse perspectives on its risks and benefits.

a) microbiome

The term “microbiome” refers to the complex community of microorganisms that live in a particular environment. These microorganisms include bacteria, fungi, viruses, and other microbes. Most commonly, when people refer to the microbiome, they are talking about the community of microbes living in and on the bodies of animals, including humans. However, microbiomes are also crucial to other environments, such as soil, water, and plants.

Human Microbiome

In humans, the microbiome plays several essential roles in maintaining health and well-being:

1. Digestion: Gut bacteria help break down complex carbohydrates and fibers that the human body cannot digest on its own, aiding in the overall digestive process.
2. Immune System Development and Function: The microbiome helps regulate the immune system. Exposure to diverse microbes, especially early in life, helps develop a more robust immune response. Microbes also protect against pathogens by competing for resources and space in the body.
3. Synthesis of Vitamins and Other Nutrients: Some gut bacteria are capable of synthesizing essential vitamins like vitamin K and some B vitamins.
4. Influence on Behavior and Mental Health: Emerging research suggests that the gut microbiome can affect the central nervous system, potentially influencing mood, behavior, and cognitive functions. This connection is often referred to as the “gut-brain axis.”

Environmental Microbiomes

In other environments, such as soil or aquatic ecosystems, microbiomes contribute to nutrient cycling, decomposition, and the overall health of the ecosystem. For instance:

1. Soil Microbiome: Microbes in the soil decompose organic matter, recycle nutrients, and support plant growth through nitrogen fixation and the breakdown of organic materials.
2. Aquatic Microbiome: In oceans and freshwater systems, microbes play a pivotal role in nutrient cycling, such as carbon and nitrogen cycling, and are integral to the food chain.

Microbiome Research and Applications

Research into microbiomes has expanded significantly with advances in genomic and bioinformatic technologies, which allow scientists to study these communities at the molecular level without needing to culture the organisms in the lab. This research has profound implications for medicine, agriculture, and environmental management, influencing everything from personalized medicine and probiotic treatments to sustainable farming practices and bioremediation strategies.

Understanding and manipulating microbiomes is a rapidly growing field that holds promise for addressing a wide range of health, environmental, and industrial challenges.

b) Lactobacillus 

is a significant group of gram-positive, non-spore-forming bacteria commonly found in various natural environments, including the human gastrointestinal tract, mouth, and vagina, as well as in fermented foods like yogurt, cheese, and sauerkraut. These bacteria are rod-shaped, typically facultative anaerobes, and are known for their role in the lactic acid fermentation process, where they convert sugars into lactic acid. This activity is crucial in the food industry for the preservation and flavor enhancement of fermented products.

Lactose intolerance is a common digestive disorder where the body is unable to digest lactose, a type of sugar mainly found in milk and dairy products. This condition stems from a deficiency in lactase, the enzyme responsible for breaking down lactose in the digestive system.

When people with lactose intolerance consume dairy products, they often experience symptoms such as bloating, diarrhea, gas, and abdominal pain. These symptoms occur because the undigested lactose moves through the intestines and is fermented by bacteria, producing gas and drawing water into the colon.

There are several types of lactose intolerance:

1. Primary lactose intolerance: The most common form, which results from a natural decline in lactase production as individuals age. This decline is genetically programmed and varies widely among populations.
2. Secondary lactose intolerance: This form is due to conditions that injure the small intestine, such as infection, celiac disease, inflammatory bowel disease, or other diseases that impair the production of lactase.
3. Developmental lactose intolerance: This occurs in premature babies whose lactase levels are not yet fully developed. It usually improves as the infants mature.
4. Congenital lactose intolerance: A very rare form, where babies are born with little or no lactase activity due to a genetic mutation. This condition requires lifelong dietary adjustments.

c) bacteria cell structure

How do bacteria multiply?

Bacteria primarily multiply through a process called binary fission, which is a form of asexual reproduction. Here’s how it works step by step:

1. Cell Growth: The bacterial cell grows and increases its overall size. During this phase, it also replicates its genetic material, ensuring that each new cell will have a complete copy of the bacterial DNA.
2. DNA Replication: The single, circular DNA molecule that most bacteria have begins to replicate. The DNA replication is bidirectional, starting from a specific location called the origin of replication, and continues until the entire genome is duplicated.

1. Cytokinesis: After DNA replication, the cell prepares to divide. A cell structure called the septum begins to form in the center of the cell. This septum grows inwards and eventually bisects the cell, although it may not be exactly at the center, depending on the species and environmental conditions.
2. Cell Division: The septum fully develops and divides the cell into two genetically identical daughter cells. Each cell will have a complete copy of the bacterial DNA and roughly half of the cytoplasmic content of the original cell.
3. Separation: The newly formed daughter cells may separate completely from each other or remain attached, forming structures like chains or clusters, depending on the species.

This process of binary fission can occur rapidly under optimal conditions. Some bacteria can divide every 20 to 30 minutes, potentially leading to exponential growth where the number of bacteria doubles with each generation. Environmental factors such as nutrient availability, temperature, and the presence of toxic substances can influence the rate of bacterial growth and division.

good bacteria vs bad bacteria

The distinction between “good” bacteria and “bad” bacteria is central to understanding how microbes affect our health and the environment.

Good Bacteria:

These beneficial microbes play essential roles in various processes vital for our health and ecological balance. Here are some key functions of good bacteria:

1. Digestive Health: In the human gut, good bacteria aid in digestion, help synthesize vitamins (like B and K), and boost immune function by protecting against pathogens.
2. Fermentation: Beneficial bacteria are used in the fermentation of foods like yogurt, cheese, and sauerkraut, contributing to food preservation and enhanced nutritional value.
3. Biodegradation: Certain bacteria are employed to break down pollutants and waste materials in the environment, aiding in natural recycling and pollution control.
4. Agriculture: Some bacteria promote plant growth by fixing nitrogen, decomposing organic material, and enhancing soil fertility.

Bad Bacteria:

Conversely, bad bacteria are those that can cause diseases and infections in humans, animals, and plants. Some prominent impacts include:

1. Infections and Diseases: Pathogenic bacteria can cause a wide range of illnesses, from mild skin infections to serious conditions like pneumonia, meningitis, and foodborne illnesses.
2. Food Spoilage: Certain bacteria are responsible for food spoilage, leading to economic loss and health risks from consuming spoiled products.
3. Antibiotic Resistance: Some bacteria have developed resistance to antibiotics, making infections harder to treat and posing significant public health challenges.

The balance between good and bad bacteria is crucial. Disruptions to this balance, such as through the overuse of antibiotics or poor dietary habits, can lead to health issues like antibiotic resistance and diminished gut health. Understanding and managing this microbial balance is a key focus in medicine, environmental science, and agriculture.

d) Plasmids

play a crucial role in synthetic biology, particularly in the engineering and manipulation of biological systems, including the creation of synthetic life forms. A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a bacterial cell’s chromosomal DNA and can replicate independently.

Here’s how plasmids contribute to synthetic biology:

1. Gene Cloning and Expression: Plasmids are used as vectors to insert foreign genes into bacteria. By inserting a gene of interest into a plasmid and then introducing that plasmid into a bacterial cell, researchers can get the bacteria to express the gene and produce the protein it codes for. This is fundamental for producing useful biological products like insulin, hormones, enzymes, and vaccines.
2. Genetic Engineering: Plasmids can be engineered to contain multiple synthetic genes, regulatory elements, and other genetic sequences. These modified plasmids can be used to program bacteria or other host cells to perform specific functions, such as producing complex molecules or degrading pollutants.
3. Creation of Novel Genetic Pathways: In synthetic biology, plasmids can be used to construct entirely new genetic circuits and pathways that do not exist in nature. By combining genetic components in new ways, scientists can create cells with customized behaviors, such as sensing environmental toxins or producing new types of biofuels.
4. CRISPR-Cas9 and Genome Editing: Plasmids are often used to deliver the CRISPR-Cas9 gene-editing system into cells. The plasmid typically contains the Cas9 gene and a guide RNA that directs the Cas9 protein to a specific location in the genome, where it makes a cut, allowing scientists to edit the DNA at that location.
5. Synthetic Cell Creation: In more advanced applications, researchers are working on creating synthetic cells from the ground up. Plasmids are used to introduce essential life-supporting genes and functions into empty lipid vesicles or minimal cell structures to create cell-like systems that exhibit life-like properties.

Through the manipulation and use of plasmids, synthetic biology aims to redesign natural biological systems for useful purposes and create new forms of life that can perform tasks beyond the capabilities of natural organisms. This approach holds tremendous potential for advancements in medicine, environmental science, and biotechnology.

synthetic microplasma

Using bacteria to detect environmental pollutants like mercury

example of application of synthetic biology and microbial biotechnology.

Researchers have engineered bacteria to become biosensors that can specifically detect and signal the presence of mercury in the environment. 

1. Genetic Engineering: Scientists start by genetically modifying bacteria to incorporate a mercury-responsive gene. This gene is often linked to a reporter system. Commonly, these genes are derived from bacterial operons that naturally respond to mercury, such as the mer operon, which confers mercury resistance.
2. Mer Operon: The mer operon includes genes that encode enzymes capable of detoxifying mercury. In engineered bacteria, parts of this operon are used to create a system where the presence of mercury activates these genes.
3. Reporter Gene: The activation of the mercury-responsive genes is coupled with the expression of a reporter gene. This gene might encode for a fluorescent protein, a bioluminescent enzyme, or another easily detectable marker. For example, when mercury is present, the bacteria might start to fluoresce or change color, indicating contamination.
4. Detection: These genetically engineered bacteria can be placed in environments suspected of mercury contamination, such as water bodies or soil samples. When mercury is present, it binds to specific receptors or enzymes encoded by the introduced genes, triggering the reporter system.
5. Applications: This approach allows for real-time, on-site detection of mercury and other heavy metals, providing a cost-effective and efficient means of monitoring environmental pollution. These biosensors are particularly useful in remote or resource-limited areas where traditional laboratory testing is not feasible.
6. Advantages and Limitations: The use of bacterial biosensors offers several advantages, including high sensitivity to specific pollutants and the ability to monitor environments continuously. However, these systems also face challenges such as maintaining the viability of bacteria in diverse environmental conditions and ensuring the specificity and accuracy of the detection under field conditions.

Overall, the use of engineered bacteria as biosensors represents a promising area in environmental monitoring, combining the natural capabilities of microbes with advanced genetic engineering techniques to tackle pollution detection and remediation.

Escherichia coli

E. coli, is a rod-shaped, gram-negative bacterium that is frequently found in the lower intestine of warm-blooded organisms. Most strains of E. coli are harmless and are actually an important part of a healthy human intestinal tract. However, some strains can cause serious food poisoning and other illnesses, such as urinary tract infections and neonatal meningitis.

E. coli is widely used in genetic research and biotechnology because it is easy to grow and its genetics are well understood. It serves as a fundamental model organism and is also used in the production of recombinant proteins and other bioproducts. In pathology, certain pathogenic strains of E. coli, such as O157:H7, are known for causing outbreaks of foodborne illness, typically associated with contaminated undercooked beef, raw milk, or fresh produce.

Cloning

 can be conducted in several different contexts, including molecular cloning, cellular cloning, and organismal cloning. Both in vitro (in a controlled environment outside a living organism) and in vivo (within a living organism) methods are used depending on the purpose and scale of the cloning.

molecular and cellular cloning processes commonly used in laboratories.

Molecular Cloning (In Vitro)

Molecular cloning involves the replication of a specific DNA sequence within a host organism, typically bacteria. This is a fundamental technique in genetic engineering used to amplify DNA sequences and produce recombinant proteins. Here are the basic steps:

1. DNA Extraction: The DNA containing the gene of interest is extracted from the organism.
2. Gene Insertion: The extracted DNA is cut with restriction enzymes and inserted into a plasmid vector that has been cut with the same enzymes, creating recombinant DNA through the use of DNA ligase.
3. Transformation: The recombinant plasmid is introduced into a host cell, typically E. coli, through a process called transformation, where cells take up the plasmid from their environment.
4. Selection: Cells that have successfully taken up the plasmid are selected using antibiotic resistance markers included in the plasmid. Only the cells that grow in the presence of the antibiotic are those that have incorporated the plasmid.
5. Cloning and Expression: The selected cells are cultured to multiply the DNA and, if the plasmid is designed for expression, to produce the protein encoded by the inserted gene.

Cellular Cloning (In Vitro)

Cellular cloning, often referred to as cell culture cloning, involves growing cells from a single cell to produce a genetically uniform population. This is important for research ensuring that all cells in a sample are identical.

1. Isolation: Cells from a multicellular organism, tissue samples, or existing cell lines are isolated.
2. Culture: The isolated cells are placed in a nutrient-rich culture medium, which supports cell growth and division.
3. Subculturing: As cells proliferate, they are routinely divided and transferred into new culture vessels to maintain optimal growth conditions and avoid over-confluence.
4. Screening: Cells may be screened for desired traits or genetic markers, especially if the cloning aims to identify cells with specific genetic modifications or properties.

In Vivo Cloning

In vivo cloning typically refers to the production of genetically identical organisms. This process includes techniques like reproductive cloning in animals, which involves transferring genetic material from the nucleus of a donor adult cell to an egg cell from which the nucleus has been removed:

1. Nuclear Transfer: The nucleus of a mature but unfertilized egg is removed and replaced with a nucleus from a donor adult somatic cell.
2. Activation and Development: The reconstructed egg is stimulated with an electric pulse or chemical trigger to start dividing and developing into an embryo.
3. Implantation: The developing embryo is then implanted into a surrogate mother where it continues to develop until birth.
4. Birth of Clone: The surrogate mother gives birth to a genetically identical clone of the donor organism.

Each of these cloning methods serves different scientific, medical, and research purposes, from studying genetic diseases in homogeneous cell populations to producing high-value pharmaceuticals or exploring genetic conservation and reproduction in animal models.

biosensor experiment: Creating a functional test for milk using Escherichia coli BL21 competent cells

Creating a functional test for milk using Escherichia coli BL21 competent cells requires several steps: making the BL21 cells competent, transforming them with a plasmid that allows for the detection of a specific substance in milk (like lactose or a contaminant), and then conducting the experiment. Here, I’ll detail a basic procedure, focusing on using a lactose-detection plasmid as an example.

Step 1: Making E. coli BL21 Competent Cells

Competent cells are able to take up foreign DNA from their environment. Here’s how to make BL21 cells competent:

Materials:

• E. coli BL21 strain
• LB medium (Lysogeny Broth)
• Calcium chloride (CaCl₂) solution (100 mM)
• Ice
• Sterile tubes and pipettes
• Centrifuge

Procedure:

1. Cultivate Cells:
   • Inoculate a fresh colony of BL21 into LB medium and grow overnight at 37°C with shaking.
2. Chill and Harvest:
   • Chill the culture on ice for 10-15 minutes.
   • Centrifuge at 4,000 x g for 10 minutes at 4°C to pellet the cells.
   • Decant the supernatant carefully.
3. Resuspend in CaCl₂:
   • Gently resuspend the pellet in cold 100 mM CaCl₂ solution.
   • Leave on ice for 30 minutes.
4. Second Centrifugation:
   • Centrifuge again under the same conditions to pellet the cells.
   • Remove the supernatant and resuspend the pellet in a smaller volume of cold 100 mM CaCl₂.
5. Aliquot and Freeze:
   • Aliquot the cell suspension into pre-chilled sterile microcentrifuge tubes.
   • Freeze immediately in a -80°C freezer for storage.

Step 2: Transformation of Competent Cells

Materials:

• Prepared competent BL21 cells
• Plasmid DNA (e.g., a plasmid encoding a lactose biosensor or reporter)
• LB agar plates with appropriate antibiotic
• Heat block or water bath set at 42°C
• Ice

Procedure:

1. Thaw Cells:
   • Thaw competent cells on ice.
2. Add DNA:
   • Add 1-10 ng of plasmid DNA to the cells and gently mix by flicking the tube.
3. Heat Shock:
   • Place the tubes in a 42°C water bath for 45-60 seconds for heat shock.
   • Immediately return the tubes to ice for 2 minutes to stabilize.
4. Recovery:
   • Add 1 mL of LB medium (without antibiotic) and incubate at 37°C for 1 hour with shaking for recovery.
5. Plate Cells:
   • Spread 100-200 µL of the transformation mix on pre-warmed LB agar plates containing the appropriate antibiotic.
   • Incubate overnight at 37°C.

Step 3: Testing Milk

Materials:

• Transformed BL21 cells grown on plates
• Milk sample
• Sterile pipettes and culture tubes

Procedure:

1. Prepare Inoculum:
   • Pick a colony from the transformation plate and inoculate it into LB broth containing the appropriate antibiotic.
   • Grow to mid-log phase.
2. Inoculate Milk:
   • Add a measured amount of the bacterial culture to a small volume of milk.
   • Incubate at 37°C with shaking.
3. Observe Results:
   • Observe any changes or signals from the biosensor (such as fluorescence, color change, etc.), depending on the design of the genetic construct in the plasmid.

II. Transgenic art

Transgenic art refers to a form of contemporary art that uses genetic engineering to create unique biological artworks. This genre fundamentally involves the manipulation of the genetic material of living organisms to explore issues surrounding biotechnology, ethics, boundaries between species, and the relationship between humans and other life forms.

Origins and Key Concepts

Transgenic art was pioneered in the late 1990s by Brazilian-American artist Eduardo Kac.  His work “GFP Bunny” is perhaps the most famous example. It involved the creation of a genetically modified rabbit named Alba that glowed green under blue light. This effect was achieved by incorporating a jellyfish gene for green fluorescent protein (GFP) into the rabbit’s genome. Kac’s aim was to raise public awareness and debate concerning the genetic manipulation of animals and the possible ramifications of biotechnological advancements.

Techniques Used

Transgenic art typically involves:

• Genetic Engineering: Artists collaborate with scientists to alter the genetic structure of living organisms, incorporating genes from different species to express new traits, such as fluorescence or altered coloration.
• CRISPR/Cas9: Recent advancements in gene editing, particularly CRISPR technology, have provided artists and scientists with more precise tools to manipulate the genomes of living organisms for artistic purposes.

Ethical and Social Implications

Transgenic art opens up numerous ethical and social questions:

• Ethical Treatment of Animals: The welfare of genetically modified organisms is a critical issue. The creation of transgenic animals raises concerns about potential suffering or health issues that might arise from genetic modifications.
• Impact on Nature and Biodiversity: There are concerns about the impact of releasing genetically modified organisms into the wild, where they could potentially disrupt local ecosystems or breed with wild populations.
• Public Perception of Science: By blending the boundaries between artistic expression and scientific practice, transgenic art influences public perceptions of genetic engineering, potentially demystifying or, conversely, vilifying this branch of science.

Examples of Transgenic Art

Beyond “GFP Bunny”, other examples include: 

• “Genesis” by Eduardo Kac:    Kac created an artwork where an artificial gene sequence, derived from a biblical verse, was translated into Morse code and then converted into DNA base pairs and inserted into bacteria. When viewers triggered a UV light, the bacteria expressed the gene, creating a glowing effect.

• Agnes Meyer-Brandis’ “Moon Goose Colony”: This project explores the boundaries of species by linking lunar exploration with the migration patterns of geese, creating a narrative that suggests genetic adaptation for life on the moon.

. Edward Steichen’s Delphiniums:

Edward Steichen, renowned for his contributions to photography, created a significant body of work that also included an impressive horticultural achievement with his Delphiniums. These flowers were not only a personal passion but also became the subject of one of his most famous exhibitions. In the early 1930s, Steichen focused his energies on breeding delphiniums, aiming to create new varieties with enhanced qualities. His efforts culminated in an exhibition titled “Delphiniums,” held at the Museum of Modern Art in New York in 1936, marking a rare instance of living plants being the primary subject of an art exhibition.

The exhibition showcased Steichen’s skill and meticulous care in the cultivation and hybridization of these flowers, which were noted for their vibrant colors and impressive stature. This work blended Steichen’s artistic vision with his horticultural expertise, presenting the delphiniums as both scientific achievements and objects of aesthetic beauty. The exhibition was highly acclaimed and is remembered as a landmark moment that illustrated the convergence of art, science, and nature through the lens of a master photographer and gardener.

The Cosmopolitan Chicken Project (CCP, 1999) is a global, transdisciplinary and transtemporal examination of the themes of biocultural diversity and identity through the interplay of art, science and beauty. In the CCP, artist Koen Vanmechelen crossbreeds chicken breeds from different countries. His ultimate goal is the creation of a Cosmopolitan Chicken carrying the genes of all the planet’s chicken breeds. Much more than a mere domesticated animal, the chicken is art in itself. It serves as a metaphor for the human animal and its relationship with the biological and cultural diversity of the planet.

Thomas Feuerstein  is an Austrian contemporary artist. His works and projects are realized in different media. They include sculptures, installations, environments, objects, drawings, paintings, radio plays as well net art and BioArt. Feuerstein’s work is known for growth and transience, processes of transformation, biological metabolism and entropy.

^{foto: Thomas Feuerstein, Installation view Frankfurter Kunstverein 2021 with the works “Hydra”, “Green Hydra” and “Green Blood” (2021) , With kind support of Muffathalle Münich, Photo: Norbert Miguletz, ©Frankfurter Kunstverein, Courtesy: the artist and Galerie Elisabeth und Klaus Thoman}

LAURA CINTI C-LAB :

^{transgenic organisms}

 

Špela Petrič

Špela Petrič is a new media artist with a background in the natural sciences. Her artistic practice combines biomedia and performativity.

Future Directions

The future of transgenic art might involve more complex genetic modifications and could potentially include the creation of entirely new forms of life. As biotechnological tools become more accessible and refined, the scope of transgenic art will likely expand, continuously challenging our ethical frameworks and artistic boundaries.

Transgenic art remains a controversial and thought-provoking intersection of art and biotechnology, questioning the roles and responsibilities of artists and scientists in the genetic age. It serves as a catalyst for dialogue about the future of life on Earth and beyond, exploring how far humanity should go in its quest to manipulate the very fabric of life.

III. DIY GENETIC MODIFICATION

with Kas Houthuijs, channeled by Marteen Smith

GFP green fluorescent

Green Fluorescent Protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Initially discovered in the jellyfish Aequorea victoria in 1962 by Osamu Shimomura, GFP has since become an invaluable tool in science, particularly in molecular and cellular biology, due to its ability to act as a biological marker.

Characteristics and Mechanism:

GFP absorbs blue light (maximum at 395 nm) and emits green light (peak at 509 nm). The fluorescent properties of GFP are due to a serine, tyrosine, and glycine sequence in its structure that forms a chromophore inside the protein. This chromophore is the part of the molecule responsible for its fluorescence and is formed in a post-translational modification that involves the cyclization and oxidation of these amino acids.

Uses in Research:

1. Gene Expression and Protein Localization: GFP can be fused to a protein of interest, and this fusion protein can be visualized in live cells or tissues using fluorescence microscopy. This allows researchers to observe where and when proteins are expressed in living organisms.
2. Reporter Gene: GFP is widely used as a reporter gene. If the GFP gene is placed under the control of a promoter from another gene, its expression can indicate the activity of the promoter, thereby providing insights into gene expression patterns and regulation.
3. Tracking Cell Development and Movement: Scientists use GFP to track cells during development, migration, or other biological processes in real-time in living organisms.
4. Biotechnological Tool: GFP has applications in biotechnology, including its use in biosensors where it acts as a signal for the presence of specific molecules.

Advancements:

The discovery and development of GFP have led to several variants with different spectral properties, such as enhanced GFP (eGFP), which has been optimized for brighter fluorescence and greater expression in mammalian cells. Other variants cover a spectrum of colors, enabling researchers to label multiple proteins or cellular structures differently within the same cell.

Recognition:

The importance of GFP in science was highlighted by the award of the Nobel Prize in Chemistry in 2008 to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for the discovery and development of GFP.

Overall, GFP remains a crucial and versatile tool in biological and medical research, providing a visual means to track and understand molecular and cellular processes.

DNA-RNA-PR

Conventional Breeding and Genetic Modification are two techniques used to alter the genetic makeup of plants and animals, but they differ significantly in methods, outcomes, and applications.

Conventional Breeding

Conventional breeding, or selective breeding, is a centuries-old method where plants or animals are selectively bred to enhance desirable traits such as yield, quality, and resistance to pests and diseases. This process involves:

• Selection: Choosing parent organisms with specific desirable traits.
• Crossbreeding: Mating these selected parents to produce offspring, which are then evaluated for desired characteristics.
• Backcrossing: Repeatedly crossing offspring with one of the parents or other related organisms to stabilize the appearance of desired traits over generations.

Conventional breeding relies on natural genetic variation and recombination. It is generally slower and may involve a degree of uncertainty as it can bring along unwanted traits due to the mixing of a large number of genes.

Genetic Modification (GM)

Genetic modification, also known as genetic engineering, is a modern technology that involves directly altering the DNA of an organism at the molecular level. This process includes:

• Gene Splicing: Scientists identify and isolate a gene responsible for a desired trait in one organism (which could be a plant, animal, or microbe) and insert it into the DNA of the organism to be modified.
• Transformation: The new DNA (often carried by a vector like a plasmid) is introduced into the target organism’s cells, integrating into the genome to express the desired trait.
• Regeneration and Selection: Modified cells are grown in culture to develop into mature plants or animals, and successful modifications are selected for further development.

Genetic modification can introduce new traits much more rapidly and precisely than conventional breeding. GM can also transcend natural species barriers, introducing genes from any species into another, which cannot be achieved with traditional breeding methods.

Key Differences

• Precision: Genetic modification offers more precision as specific genes can be targeted and modified, whereas conventional breeding involves more random, broad-scale genetic exchanges.
• Speed: GM can achieve desired genetic traits in a much shorter time frame compared to the multiple growing seasons often required for conventional breeding.
• Species Barriers: GM can overcome natural reproductive barriers, allowing for the introduction of traits from unrelated species (like bacterial genes in plants).

Ethical and Regulatory Considerations

Both methods raise different ethical and regulatory concerns. Conventional breeding is generally accepted and viewed as “natural,” although it can also raise concerns when it involves extreme traits in animals (like in some dog breeds). GM, on the other hand, faces significant scrutiny regarding biosafety, potential environmental impacts, and food safety, leading to stringent regulations in many countries.

Both techniques are crucial in addressing challenges such as food security, climate change adaptation, and sustainable agriculture, each playing a role according to the specific needs and circumstances of their application.

Genetic Engineering

Transforming E. coli with pGLO Plasmid

pGLO Plasmid Map and Resources (download from the link)

Transforming E. coli bacteria with the pGLO plasmid is a standard lab procedure used in genetics and biotechnology education. The pGLO plasmid contains a gene for green fluorescent protein (GFP), originally extracted from a jellyfish, which makes transformed cells glow green under UV light. The plasmid also contains a gene for resistance to the antibiotic ampicillin, which allows for the selection of transformed cells. Here’s a step-by-step guide to the transformation process:

Materials Needed

• Competent E. coli cells (prepared to take up DNA)
• pGLO plasmid DNA
• LB nutrient agar plates, some containing ampicillin, some with both ampicillin and arabinose (a sugar that triggers the GFP gene)
• Calcium chloride (CaCl₂) solution for transformation buffer
• Ice and ice bucket
• Heat block or water bath set to 42°C
• Sterile microcentrifuge tubes
• Sterile inoculation loops or pipette tips
• Incubator set to 37°C

Steps for pGLO Transformation

1. Thaw Competent Cells

• Place the vial of competent E. coli cells on ice and allow them to thaw slowly.

2. Add pGLO Plasmid DNA

• Label one microcentrifuge tube “pGLO” and another “control”.
• Transfer 200 µl of competent cells into each tube.
• Add 10 µl of pGLO plasmid solution to the “pGLO” tube. Do not add plasmid to the control tube.

3. Incubate on Ice

• Place both tubes on ice for 10 minutes. This step helps the cells to become more permeable to DNA uptake.

4. Heat Shock

• Heat shock the cells by placing the tubes into a water bath or heat block at 42°C for exactly 50 seconds. This sudden increase in temperature creates a thermal imbalance across the cell membrane, encouraging the cells to take up the plasmid.
• Immediately return the tubes to ice for another 2 minutes to help cells recover and close their membranes.

5. Recovery

• Add 250 µl of LB broth (without antibiotic) to each tube to provide nutrients for the cells.
• Incubate the tubes at 37°C for 60 minutes. This allows the bacteria to recover and begin expressing the antibiotic resistance gene.

6. Plate the Cells

• Using a sterile loop or pipette, spread 100-150 µl from each tube onto the prepared LB agar plates:
  • Spread the “control” tube cells on an LB plate without ampicillin.
  • Spread the “pGLO” tube cells on plates with ampicillin, and with ampicillin plus arabinose.
• This step uses the ampicillin to select for cells that have taken up the pGLO plasmid, and arabinose to induce expression of the GFP.

7. Incubate the Plates

• Incubate the plates upside down in a 37°C incubator overnight.

8. Observe Results

• Check the plates for growth after 24 hours:
  • The “control” plate should show growth only on the LB plate without ampicillin.
  • The “pGLO + Amp” plate should show growth if transformation was successful.
  • The “pGLO + Amp + Ara” plate should not only show growth but also fluorescent colonies under UV light if the GFP gene is expressed.

Conclusion

This transformation procedure allows you to insert a foreign plasmid into E. coli and have the bacteria express new genes. It’s a fundamental technique for genetic engineering studies, demonstrating how genes can be manipulated to express specific traits such as antibiotic resistance and fluorescence.